KR102085605B1 - Fusion proteins for treating metabolic disorders - Google Patents

Abstract

The present invention relates to the identification of fusion proteins comprising polypeptides and protein variants of fibroblast growth factor 21 (FGF21) having improved pharmaceutical properties. Also disclosed are methods of treating FGF21-associated disorders, including metabolic conditions.

Description

FUSION PROTEINS FOR TREATING METABOLIC DISORDERS}

The present invention relates to novel fusion proteins comprising fibroblast growth factor 21 (FGF21), which are known to improve metabolic profiles in a subject to be administered.

The fibroblast growth factor (FGF) family is characterized by 22 genetically distinct homologous ligands, which are divided into seven subfamily. FGF-21 is most closely associated with FGF-19 and FGF-23 and together with them form a subfamily. This FGF subfamily regulates various physiological processes that are not common to typical FGFs: energy and bile acid homeostasis, glucose and lipid metabolism, and phosphate as well as vitamin D homeostasis. In addition, unlike other FGFs, this subfamily acts in an endocrine fashion (Moore, DD (2007) Science 316, 1436-8) (Beenken et al. (2009) Nature Reviews Drug Discovery 8, 235)]).

FGF21 is a 209 amino acid polypeptide containing 28 amino acid leader sequences (SEQ ID NO: 5). Human FGF21 has about 79% amino acid identity to mouse FGF21 and about 80% amino acid identity to rat FGF21. Fibroblast growth factor 21 (FGF21) has been described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of lung, bronchial or alveolar cell function (Nishimura et al. (2000) Biochimica et Biophysica Acta). 1492: 203-206; and Patent Publication WO01 / 36640; and Patent Publication WO01 / 18172. FGF-21 activates FGF receptors and downstream signaling molecules including FRS2a and ERK, but no direct interaction of FGFR with FGF-21 was detected. Research has identified β-klotho, a highly expressed cofactor in the liver, adipocytes and pancreas, as a determinant of cellular responses to FGF-21 and as a cofactor for mediating FGF-21 signaling through FGFR. (Kurosu, H. et al. (2007) J Biol Chem 282, 26687-95). FGF21 is a potent agonist of FGFR1 (IIIc), FGFR2 (IIIc) and FGFR3 (IIIc) β-cloto signaling complexes.

FGF-21 has been shown to induce insulin-independent glucose uptake. FGF-21 has also been shown to improve hyperglycemia in various diabetic rodent models. In addition, transgenic mice overexpressing FGF-21 have been shown to be resistant to dietary-induced metabolic abnormalities, and demonstrated reduced weight and fat mass, and enhancement of insulin sensitivity (Badman, MK et al. (2007). Cell Metab 5, 426-37]. Administration of FGF-21 to diabetic non-human primates resulted in a decrease in fasting plasma glucose, triglycerides, insulin and glucagon levels, leading to significant improvements in lipoprotein profiles, including an almost 80% increase in HDL cholesterol. Kharitonenkov, A. et al. (2007) Endocrinology 148, 774-81]. Recent studies investigating the molecular mechanism of FGF21 action have identified FGF21 as an important endocrine hormone that helps regulate adaptation to fasting conditions (Badman et al. (2009) Endocrinology 150, 4931 (Inagaki et al. (2007) Cell Metabolism 5, 415)]). This provides a previously lost link downstream of PPARα that allows the liver to communicate with the rest of the body in regulating the biology of energy homeostasis (Galman et al. (2008) Cell Metabolism 8, 169 (Lundasen et al. (2007) Biochemical and Biophysical Research Communications 360, 437)]).

FGF21 regulates adipocyte homeostasis through activation of the AMPK / SIRT1 / PGC1α pathway, which inhibits PPARγ expression and increases mitochondrial function (Chau et al. (2010) PNAS 107, 12553). FGF21 also increases glucose uptake by skeletal muscle as measured in cultured human root canals and isolated mouse tissues. FGF21 treatment of rodent islets leads to improved function and survival through activation of the ERK1 / 2 and Akt pathways (Wente et al. (2006) Diabetes 55, 2470). FGF21 treatment also causes altered gene expression for lipidogenesis and fatty acid oxidase in rodent livers, possibly via HNF4α and Foxa2 signaling.

A problem associated with the direct use of FGF-21 as a biotherapeutic agent is that its half-life is very short (Kharitonenkov, A. et al. (2005) Journal of Clinical Investigation 115: 1627-1635). In mice, the half-life of human FGF21 is 0.5-1 hour, and in cynomolgus monkeys, the half-life is 2-3 hours. FGF21 can also be used as a versatile sterile pharmaceutical formulation. However, preservatives, ie m-cresols, have been determined to have deleterious effects on their stability under these conditions.

In the development of FGF21 protein for use as a therapeutic in the treatment of type 1 and type 2 diabetes and other metabolic conditions, increased half-life and stability would be desirable. FGF21 protein with enhanced half-life and stability will allow patients to receive less frequent protein administration. Clearly, there is a need to develop stable aqueous protein formulations for therapeutic protein FGF21.

In addition, a significant problem in the development of FGF21 as a protein pharmaceutical is to overcome its physical and chemical instability. Compositional diversity and characteristics of proteins define specific behaviors such as folding, conformational stability, and unfolding / denaturation. This feature should be addressed when targeting protein stabilization in the development of pharmaceutical formulations using aqueous protein solutions (Wang, W., Int. J. of Pharmaceutics, 18, (1999)). A desirable effect of stabilizing a therapeutic protein of interest, eg, a protein of the present invention, is to improve protein stability and reduce protein aggregation by increasing resistance to proteolytic and enzymatic degradation.

Summary of the Invention

The present invention comprises fibroblast growth factor 21 (FGF21) and has, under pharmaceutical formulation conditions, improved pharmaceutical properties over wild type FGF21 and its variants, for example, more stable, and the ability to improve metabolic parameters for subjects to be administered. It is directed to the identification of novel fusion proteins that possess a low molecular weight, are less susceptible to proteolytic and enzymatic degradation, and are less likely to aggregate and form complexes. Fusion proteins of the invention include truncations, mutations and mutations of FGF21.

FGF21-associated disorders, as well as other metabolic, endocrine and cardiovascular disorders such as obesity, type 1 and type 2 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), Insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, Also disclosed are methods for treating gastric palsy, disorders associated with severe inactivating mutations in insulin receptors, and other metabolic disorders, and methods for reducing mortality and morbidity in critically ill patients.

The fusion proteins of the invention may be used in injectable form once weekly, alone or in combination with oral antidiabetic agents, which will improve blood sugar control, body weight and lipid profiles of type 1 and type 2 diabetic patients. Proteins can also be used for the treatment of obesity or other FGF21-associated conditions.

The fusion proteins of the invention are more stable than wild type FGF21 under pharmaceutical formulation conditions, are less susceptible to proteolytic and enzymatic degradation, and are less likely to aggregate and form complexes, for example administration of wild type FGF21. Overcome significant challenges of physical instability associated with protein therapeutics, including.

In a first aspect, the invention provides a fibroblast growth factor 21 (FGF21) fusion protein comprising one or more of the sequences listed in Table 1 and further described herein. The FGF21 sequence listed in Table 1 has a wild type FGF21 sequence, for example NCBI Ref. No. NP_061986.1, and can be found in a granted wild type FGF21 such as, for example, US 6,716,626B1 assigned to Chiron Corporation. It may be a variant of the sequence.

Such fusions can be, for example, variant FGF21 sequences, such as the sequences in Table 1 and other molecules (non-FGF21 moieties), eg IgG constant domains or fragments thereof (eg Fc regions), human serum albumin (HSA ) Or a fusion between albumin-binding polypeptide. In a preferred embodiment, the non-FGF21 portion of the molecule is an Fc region.

Another embodiment relates to a polynucleotide encoding a fusion protein of the invention, a vector containing said polynucleotide and a host cell carrying said vector.

Provided herein are methods for use in generating the fusion proteins of the invention, wherein such methods include, for example, modification of the wild type FGF21 protein, as well as site-specific incorporation of amino acids at positions of interest in the wild type FGF21 protein, as well as Other molecules such as lgG constant domains or fragments thereof (eg Fc regions), human serum albumin (HSA), or albumin-binding polypeptides and fusions between the FGF21 portion of the molecule. Such modifications and fusions enhance the biological properties of the fusion proteins of the invention relative to the wild-type version of the protein, as well as in some cases serve as points of attachment for, for example, labels and protein half-life extenders, and the surface of the solid support Acts to attach the variant to it. A related embodiment of the present invention is a method of producing a cell capable of producing the protein of the present invention and a vector containing a DNA encoding the variant and fusion.

In various embodiments, the fusion proteins of the invention disclosed herein can comprise one or more fragments of the FGF21 wild type sequence, including fragments as small as 8-12 amino acid residues in length, wherein the polypeptide lowers blood glucose in a mammal. You can. In various embodiments, the fusion proteins of the invention disclosed herein can comprise one or more variants of the FGF21 wild type sequence, eg, having one or more amino acid deletions, insertions, additions or substitutions relative to its wild type sequence.

In some embodiments, the fusion proteins of the present invention disclosed herein may comprise one or more polymers, such as polyethylene glycol (PEG), at the site where site-specific amino acid modifications have been made relative to wild type FGF21, or at the location of amino acids that share a wild type version of these proteins. Or covalently linked to polysialic acid. PEG groups are attached in a manner that does not enhance and / or inhibit the biological function of the constituent portions of the fusion proteins of the invention, eg, FGF21 protein variants. In other embodiments, polypeptides of the invention may optionally be fused to a heterologous amino acid sequence via a linker such as GS, GGGGSGGGGSGGGGS (SEQ ID NO: 6). The heterologous amino acid sequence may be an IgG constant domain or fragment thereof (eg an Fc region), human serum albumin (HSA), or albumin-binding polypeptide. Such fusion proteins disclosed herein may also form multimers.

In some embodiments, the heterologous amino acid sequence (eg HSA, Fc, etc.) is fused to the amino-terminus of the fusion protein of the invention. In other embodiments, the fusion heterologous amino acid sequence (eg HSA, Fc, etc.) is fused to the carboxyl-terminus of the fusion protein of the invention.

Another embodiment includes one or more FGF21-associated disorders such as obesity, type 2 diabetes, type 1 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance , Hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral artery disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, stomach Patients exhibiting the disorder comprising administering a therapeutically effective amount of one or more proteins of the invention or a pharmaceutical composition thereof to a patient in need of treatment for paralysis, disorders associated with inactivation mutations in the insulin receptor, and other metabolic disorders It is about how to treat it.

The invention also provides a pharmaceutical composition comprising the fusion protein of the invention disclosed herein and a pharmaceutically acceptable formulation agent. Such pharmaceutical compositions can be used in methods of treating metabolic disorders, the methods comprising administering a pharmaceutical composition of the invention to a human patient in need thereof. Non-limiting examples of treatable metabolic disorders include type 1 and type 2 diabetes and obesity.

These and other aspects of the invention will be described in the detailed description of the invention that follows.

1A-1D show that V188 has improved efficacy over V76 in an ob / ob diabetic mouse model. V188 shows superior results when administered at 1 milligram per kilogram (mpk) compared to when V76 was administered at 5 milligrams per kilogram. 1A shows postprandial plasma glucose as readout (circles represent vehicle (PBS-phosphate buffered saline), squares represent 5 mpk V76, triangles represent 1 mpk V188). 1B shows postprandial plasma insulin as readings (from left to right: vehicle, V76 at 5 mpk and V188 at 1 mpk). 1C shows body weight as reading (from left to right: vehicle, V76 at 5 mpk and V188 at 1 mpk). 1D shows hepatic lipid content as readout (from left to right: vehicle, V76 at 5 mpk and V188 at 1 mpk).
2A-2D show that V101 has improved efficacy over V76 in an ob / ob diabetic mouse model. V101 shows superior results when administered at 1 milligram per kilogram (mpk) compared to when V76 was administered at 5 milligrams per kilogram. 2A shows post-prandial plasma glucose as readout (circles represent vehicle (PBS-phosphate buffered saline), squares represent 5 mpk V76, triangles represent 1 mpk V101). 2B shows postprandial plasma insulin as readings (from left to right: vehicle, V76 at 5 mpk and V101 at 1 mpk). 2C shows body weight as reading (from left to right: vehicle, V76 at 5 mpk and V101 at 1 mpk). 2D shows hepatic lipid content as reading (from left to right: vehicle, V76 at 5 mpk and V101 at 1 mpk).
3A-3D show that V103 has improved efficacy over V76 in an ob / ob diabetic mouse model. V103 shows superior results when administered at 1 milligram per kilogram (mpk) compared to when V76 was administered at 5 milligrams per kilogram. 3A shows postprandial plasma glucose as readout (circles represent vehicle (PBS-phosphate buffered saline), squares represent 5 mpk V76, triangles represent 1 mpk V103). 3B shows postprandial plasma insulin as readings (from left to right: vehicle, V76 at 5 mpk and V103 at 1 mpk). 3C shows body weight as reading (from left to right: vehicle, V76 at 5 mpk and V103 at 1 mpk). 3D shows hepatic lipid content as reading (from left to right: vehicle, V76 at 5 mpk and V103 at 1 mpk).
4A-4D demonstrate pharmacokinetic and thermodynamic properties superior to the FGF21 fusion proteins of the art possessed by the fusion proteins of the present invention. 4A shows PCT publication WO10 as described by Fc-L (15) -FGF21 (L98R, P171G) and Fc-L (15) -FGF21 (L98R, P171G, A180E) following IV injection of the fusion in mice. Plasma concentrations of the fusion proteins of the invention at / 129600. 4B shows the pharmacokinetic properties of the fusion proteins (V101, V103 & V188) of the invention after a single IV administration in mice as assayed by anti-Fc-ELISA, in a previous study using anti-FGF21 antibody ELISA. Shown in comparison to pharmacokinetic data generated in mice for V76. 4C shows a randomized extraction test of the fusion proteins of the invention in anti-FGF21 western blot, consistent with Fc-ELISA data at 120 hours and 15 days. Samples in the blot are as follows: A represents V101, B represents V103 and C represents V188. Controls are V101 and serum. 4D demonstrates the thermodynamic stability of the fusion proteins of the present invention significantly increased compared to V76. From top to bottom, the figures show V101, V103 and V188, all of which are improved over V76 (T _m <50 ° C. (not shown)) and wild type FGF21 (T _m = 46.5 ° C. ± 0.3 (not shown)). Has a melting temperature (T _m ).

Fusion proteins of the invention represent a modified version of the full length wild type FGF21 polypeptide as known in the art. The FGF21 wild type sequence will serve as a reference sequence (SEQ ID NO: 1), for example, when a comparison between the FGF21 wild type sequence and a protein variant is required. The FGF21 wild type sequence has the NCBI reference sequence number NP_061986.1 and can be found in a granted patent such as US 6,716,626B1 assigned to Chiron Corporation, for example (SEQ ID NO: 1).

The corresponding mRNA sequence (NCBI reference SEQ ID NO: NM_019113.2) encoding the full length FGF21 polypeptide is shown below (SEQ ID NO: 2).

The mature FGF21 sequence lacks a leader sequence and also undergoes other modifications of the polypeptide, such as proteolytic processing of amino and / or carboxyl ends (with or without leader sequences), smaller polypeptide cleavage from larger precursors, N-linked And / or O-linked glycosylation, and other post-translational modifications as understood by those skilled in the art. Representative examples of mature FGF21 sequences have the following sequence (SEQ ID NO: 3, indicating amino acid positions 29-209 of the full length FGF21 protein sequence (NCBI reference SEQ ID NO: NP_061986.1)).

The corresponding cDNA sequence encoding the mature FGF21 polypeptide (SEQ ID NO: 3) is shown below (SEQ ID NO: 4).

Fusion proteins of the invention may comprise protein variants or mutants of the wild type proteins listed herein, eg, FGF21 variants. As used herein, the terms “protein variant”, “human variant”, “polypeptide or protein variant”, “variant”, “mutant”, as well as any similar term or specific version thereof (eg “FGF21 protein” Variant "," variant "," FGF21 mutant ", etc.) define a protein or polypeptide sequence comprising a naturally occurring (ie, wild-type) protein or polypeptide counterpart or a modification, truncation, or other variation of the corresponding native sequence. . “Variant FGF21” or “FGF21 mutant” is described, for example, relative to wild-type (ie naturally occurring) FGF21 protein as described herein.

Representative fusion protein sequences of the invention are listed in Table 1. The description of such fusions includes FGF21 variants, and, where applicable, linkers. Changes or substitutions used by the FGF21 variant are numbered and described for wild type FGF21. By way of example, “Variant 101 (V101)” (SEQ ID NO: 10) is an Fc-FGF21 fusion with the following substitutions made for two amino acid linkers and wild type FGF21: Q55C, A109T, G148C, K150R, P158S, P174L, S195A, P199G , G202A.

Table 1. FGF21 variant Fc fusion proteins

^* -Unless otherwise specified, note that the FGF21 wild-type sequence in the table refers to NCBI reference SEQ ID NO: NP_061986.1 (SEQ ID NO: 1). All mutations in the FGF21 moiety and the corresponding amino acid numbering of the mutations refer back to (SEQ ID NO: 1) above, rather than the full length sequence, which may also include the Fc and linker regions.

Variants or mutants used in the fusion proteins of the invention, such as those of wild type FGF21, are characterized by one or more substituted, added and / or removed amino acids relative to the wild type protein. In addition, the variants may comprise truncations of the N- and / or C-terminus relative to the wild type protein. Generally speaking, variants retain some modified structural or functional properties of wild type proteins. For example, variants may maintain enhanced or improved physical stability (eg, less hydrophobic mediated aggregation) in concentrated solutions, enhanced or improved plasma stability when incubated with plasma, or an advantageous bioactivity profile. It may have enhanced or improved bioactivity.

Acceptable amino acid substitutions and modifications that make up the difference between a portion of the fusion protein of the invention and its wild type comparison protein include, but are not limited to, one or more amino acid substitutions, including truncation by non-naturally occurring amino acid analogs, and truncation. Does not. Thus, fusion proteins of the invention (eg, fusion proteins of the invention) are site-directed mutants, truncated polypeptides, proteolysis-resistant mutants, aggregation-reducing mutants, combinatorial mutations as described herein. Sieves, and fusion proteins.

One of ordinary skill in the art of protein expression is E. For expression in E. coli, it will be appreciated that methionine or methionine-arginine sequences may be introduced at the N-terminus of any fusion protein of the invention and may be considered within the context of the invention. .

The fusion proteins of the present invention may possess increased compatibility with pharmaceutical preservatives (eg m-cresol, phenol, benzyl alcohol), and thus preserved pharmaceutical formulations that maintain the physicochemical properties and biological activity of the protein during storage. Enables the production of Thus, variants with enhanced pharmaceutical stability compared to wild type have improved physical stability in concentrated solutions under both physiological and preserved pharmaceutical formulation conditions, while maintaining biological potency. As a non-limiting example, a fusion protein of the invention may be more resistant to proteolytic and enzymatic degradation than its wild type counterpart or corresponding native sequence; May have improved stability; The probability of aggregation may be low. These terms, as used herein, are not mutually exclusive or limiting, and it is entirely possible for a given variant to have one or more modified properties of the wild type protein.

The invention also includes, for example, an FGF21 amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 3, but has desirable properties for FGF21 protein variants, such as improved potency, proteolysis-resistance, for FGF21-receptors, Particular residues that confer increased half-life or aggregate-reducing properties and combinations thereof include nucleic acid molecules encoding fusion proteins of the invention that are not further modified. That is, about 5% (alternatively 4%, alternatively 3%) in the FGF21 mutant sequence, except for residues in the FGF21 mutant sequence that have been modified to confer proteolysis-resistance, aggregation-reduction, or other properties. Alternatively 2%, alternatively 1%) of all other amino acid residues may be modified. Such FGF21 mutants retain the activity of one or more wild type FGF21 polypeptides.

The invention also encompasses nucleic acid molecules comprising a nucleotide sequence that is at least about 85% identical to, and more preferably at least about 90 to 95%, identical to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, Nucleotides encoding amino acid residues that confer resistance, aggregation-reduction, or other properties are not further modified. That is, about 15%, more preferably about 10-5%, within the mutant sequence except for nucleotides encoding residues in the modified FGF21 mutant sequence to confer proteolysis-resistance, aggregation-reduction or other properties All other nucleotides of can be modified. Such nucleic acid molecules encode proteins that retain the activity of one or more wild-type counterparts thereof.

Provided herein are methods for use in generating fusion proteins of the invention, wherein such methods are site-specific modifications and non-site-specific of wild-type versions of proteins (eg, FGF21 wild-type proteins as described herein). Modifications, such as truncation of the wild type protein, and site-specific incorporation of amino acids at positions of interest in the wild type protein. Such modifications enhance the biological properties of the fusion proteins of the invention over wild-type proteins, as well as in some cases serve as points of attachment for, for example, labels and protein half-life extenders, and provide the variants to the surface of a solid support. Acts to attach. A related embodiment of the present invention is a method of producing a cell capable of producing the fusion protein of the present invention and a vector containing a DNA encoding the variant.

In certain embodiments, such modifications, eg, site-specific modifications, are for example conjugates, eg, to extend the half-life or otherwise improve biological properties of the proteins, polypeptides and / or peptides of the invention. PEG groups are used to attach to the proteins, polypeptides and / or peptides. The technique is further described herein.

In other embodiments, such modifications, eg, site-specific modifications, are used to attach other polymer, small molecule and recombinant protein sequences that extend the half-life of the protein of the invention. One such embodiment includes the attachment of fatty acids or certain albumin binding compounds to proteins, polypeptides and / or peptides. In other embodiments, the modifications are made at specific amino acid types and can be attached to one or more sites on the protein.

In other embodiments, such modifications, eg, site-specific modifications, are attachments for the production of wild type and / or variant multimers, eg, dimers (homodimers or heterodimers) or trimers or tetramers. It is used as a means. These multimeric protein molecules may further have groups such as PEG, sugar and / or PEG-cholesterol conjugates attached or fused to other proteins, such as Fc, human serum albumin (HSA), at the amino-terminus or at the carboxy-terminus. .

In other embodiments, such site-specific modifications include site-specifically incorporated pyrrolysine, or the position of pyrrolysine analogs or non-naturally occurring amino acids (para-acetyl-Phe, para-azido-Phe). Is used to produce such proteins, polypeptides and / or peptides or with PEG, sugar and / or PEG-cholesterol conjugates, wherein the proteins allow controlled orientation and attachment of the proteins, polypeptides and / or peptides on the solid support surface. Used to ensure that the same group is attached.

In other embodiments, such site-specific modifications are used to site-specifically crosslink proteins, polypeptides and / or peptides to form hetero-oligomers including but not limited to heterodimers and heterotrimers. . In other embodiments, such site-specific modifications site-specifically crosslink proteins, polypeptides, and / or peptides to form protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates, polypeptide- Used to form peptide conjugates or peptide-peptide conjugates. In other embodiments, site specific modifications may comprise branching points that allow more than one type of molecule to be attached to a single site of a protein, polypeptide or peptide.

In other embodiments, the modifications listed herein are made in a non-site-specific manner such that the protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates, polypeptide-peptide conjugates or peptides of the invention -Peptide conjugates can be formed.

Justice

Various definitions are used throughout this specification. Most words have the meaning assigned to them by those skilled in the art. Words specifically defined below or elsewhere in this specification have the meaning as provided in the context of the present invention as a whole and as typically understood by one of ordinary skill in the art.

The term "FGF21" as used herein refers to a member of the fibroblast growth factor (FGF) protein family. The amino acid sequence of FGF21 (GenBank Accession No. NP — 061986.1) is shown as SEQ ID NO: 1 and its corresponding polynucleotide sequence is shown as SEQ ID NO: 2 (NCBI Reference SEQ ID NO: NM_019113.2). "FGF21 variants", "FGF21 mutants" and similar terms describe modified versions of the FGF21 protein, for example with constituent amino acid residues that are deleted, added, modified, or substituted.

The term "FGF21 receptor" as used herein refers to a receptor for FGF21 (Kharitonenkov, A, et al. (2008) Journal of Cellular Physiology 215: 1-7; Kurosu, Het al. (2007) JBC 282: 26687-26695; Ogawa, Yet al. (2007) PNAS 104: 7432-7437].

The term "FGF21 polypeptide" refers to a naturally occurring polypeptide expressed in humans. For the purposes of the present disclosure, the term “FGF21 polypeptide” refers to any full length FGF21 polypeptide, eg, SEQ ID NO: 1 consisting of 209 amino acid residues and encoded by the nucleotide sequence of SEQ ID NO: 2; The 28 amino acids consisting of 181 amino acid residues and at the amino-terminal end of the full length FGF21 polypeptide (ie, constituting the signal peptide) can be used interchangeably to mean any mature form of polypeptide that has been removed.

“Variant 76” as used herein is an FGF21 protein variant characterized by eight point mutations for 40 kDa branched PEG and 177 amino acid wild type proteins linked via Cys154. The synthesis of the variants is described in more detail herein, and the protein sequences are shown in Table 1 and SEQ ID NO: 9.

The term “isolated nucleic acid molecule” refers to (1) isolation from at least about 50% of proteins, lipids, carbohydrates, or other substances found in nature with the entire nucleic acid when isolated from source cells, or (2) “isolated”. Nucleic acid molecule "is naturally linked as part of a polynucleotide sequence that is not linked to all or a portion of the polynucleotide to which it is linked in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) It means a nucleic acid molecule of the invention that does not occur in. Preferably, the isolated nucleic acid molecule of the present invention is substantially free of any other contaminating nucleic acid molecule, or other contaminant found in its natural environment that inhibits its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use. I never do that.

The term "vector" is used to mean any molecule (eg, nucleic acid, plasmid or virus) used to deliver coding information to a host cell.

The term “expression vector” means a vector containing a nucleic acid sequence that is suitable for transformation of a host cell and directs and / or controls the expression of the inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing when introns are present.

The term “operably linked” is used herein to mean an arrangement of flanking sequences in which the flanking sequences so described are set up or assembled to perform their usual functions. The elements of the fusion protein may be operably linked to each other to allow the fusion protein to function as if it were a naturally occurring endogenous protein and / or to bind heterologous elements of the fusion protein in a synergistic manner.

At the nucleotide level, a fling sequence operably linked to a coding sequence can affect the replication, transcription and / or translation of the coding sequence. For example, a coding sequence is one that is operably linked to a promoter when the promoter can direct the transcription of that coding sequence. The flanking sequences need not be adjacent to the coding sequence as long as they function correctly. Thus, for example, an untranslated but transcribed intervening sequence may be present between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

The term “host cell” is used to mean a cell that can be transformed or transformed with a nucleic acid sequence and then can express a gene of interest of choice. The term includes the progeny of the parent cell, as long as the selected gene is present, whether or not the form or genetic makeup of the progeny is identical to the original parent cell.

As used herein, the term “amino acid” refers to naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimetics that function in a similar manner to naturally occurring amino acids, all of which allow D and L stereoisomeric forms In the form of such stereoisomers). Amino acids are referred to herein by their names, their commonly known three letter symbols, or the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The term “naturally occurring” when used in connection with a biological material such as a nucleic acid molecule, polypeptide, host cell, and the like, means a material found in nature and not manipulated by humans. Similarly, "non-naturally occurring" as used herein means a substance that is not found in nature or that is structurally modified or synthesized by humans. When used in the context of nucleotides, the term “naturally occurring” means base adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U). When used in connection with amino acids, the term “naturally occurring” refers to 20 common amino acids (ie, alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine ( G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine ( S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), as well as selenocysteine, pyrrolysine (Pyl or O), and pyrroline-carboxy-lysine (Pcl or Z).

Pyrrolysine (Pyl) is an amino acid found naturally in the methylamine methyltransferase of methanogenic archaea with Methanosarcina. Pyrrolysine is a lysine analog that is incorporated at the same time as translation in the in-frame UAG codon in each mRNA, which is considered the 22nd natural amino acid.

As described in at least PCT patent publication WO2010 / 48582 (filed by ILM, LLC). Attempts to biosynthesize pyrrolysine (Pyl) in E. coli resulted in the formation of “demethylated pyrrolysine” referred to herein as pyrroline-carboxy-lysine or Pcl. "Pcl" as used herein means Pcl-A or Pcl-B.

As used herein, the terms "non-natural amino acid" and "non-natural amino acid", whether identical or different, cannot be biosynthetically produced in any organism using unmodified or modified genes from any organism. It is used interchangeably to indicate amino acid structure. The term refers to an amino acid residue that is not present in a naturally occurring (wild type) FGF21 protein sequence or a sequence of the invention. These are modified but not one of the 20 naturally occurring amino acids, selenocysteine, pyrrolysine (Pyl), or pyrroline-carboxy-lysine (Pcl, for example as described in PCT Patent Publication WO2010 / 48582). Amino acid and / or amino acid analogues include, but are not limited to. Such non-natural amino acid residues may be introduced by substitution of naturally occurring amino acids and / or by insertion into a naturally occurring (wild type) FGF21 protein sequence or a sequence of the invention. In addition, non-natural amino acid residues may be incorporated to confer FGF21 molecules the ability to link a desired functional group, such as a functional moiety (eg PEG). When used in connection with amino acids, the symbol "U" shall mean "non-natural amino acids" and "non-natural amino acids" as used herein.

In addition, such “non-natural amino acids” are understood to require modified tRNA and modified tRNA synthetase (RS) for incorporation into proteins. These "selected" orthogonal tRNA / RS pairs are generated by selection methods or random or targeting mutations developed by Schultz et al. For example, pyrroline-carboxy-lysine is a "natural amino acid" in that it is biosynthetically produced by genes transferred from one organism to host cells, and incorporated into proteins using native tRNA and tRNA synthetase genes. P-aminophenylalanine (Generation of a bacterium with a 21 amino acid genetic code, Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG. J Am Chem Soc. 2003 Jan 29; 125 (4): 935-9] are "non-natural amino acids" because they are incorporated into proteins by "selected" orthogonal tRNA / tRNA synthetase pairs, even though biosynthetic.

Modified encoded amino acids are hydroxyproline, γ-carboxyglutamate, O-phosphoseline, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-amino Butyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminobutyric acid, 3-aminobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, des Mossin, 2,2'-diaminopimelic acid, 2,3-diaminoproprioric acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmocin, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, Norvaline, norleucine, ornithine, pentylglycine, pipecolic acid, and thiroline It is not. The term “amino acid” also includes naturally occurring amino acids that are metabolites in certain organisms but are not encoded by the genetic code for incorporation into proteins. Such amino acids include, but are not limited to, ornithine, D-ornithine and D-arginine.

As used herein, the term "amino acid analog" refers to a compound having the same basic chemical structure as an naturally occurring amino acid, such as hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group. Amino acid analogs include natural and unnatural amino acids that are chemically blocked reversibly or irreversibly, or chemically modified at their C-terminal carboxyl groups, their N-terminal amino groups and / or their side chain functional groups. Such analogues include methionine sulfoxide, methionine sulfone, S- (carboxymethyl) -cysteine, S- (carboxymethyl) -cysteine sulfoxide, S- (carboxymethyl) -cysteine sulfone, aspartic acid- (beta-methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and methionine methyl sulfonium.

As used herein, the term “amino acid mimetic” refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but which functions in a similar manner to naturally occurring amino acids.

The term “biologically active variant” refers to the activity of its wild type (eg naturally occurring) protein or polypeptide counterpart, such as blood glucose, HbA1c, insulin, triglycerides or cholesterol levels, regardless of the type or number of modifications introduced into the polypeptide variant. Adjust; Increase pancreatic function; Reduce liver lipid levels; Reduce weight; By any polypeptide variant used, for example, as a constituent protein of a fusion in a fusion protein of the invention, which possesses the ability to improve glucose tolerance, energy expenditure or insulin sensitivity. Polypeptide variants that possess somewhat reduced levels of activity compared to their wild-type versions can also be considered biologically active polypeptide variants. Representative non-limiting examples of biologically active polypeptide variants of the invention are FGF21 variants that retain similar or enhanced biological properties compared to wild type FGF21 after modification.

The terms “effective amount” and “therapeutically effective amount” respectively reduce one or more biological activities, such as blood glucose, insulin, triglyceride or cholesterol levels, of a wild-type polypeptide or protein counterpart; Reduce liver triglycerides or lipid levels; Reduce weight; Or an amount of the fusion protein of the invention used to support an observable level of ability to improve glucose tolerance, energy expenditure or insulin sensitivity. For example, a "therapeutically effective amount" administered to a patient who exhibits, suffers from, or is prone to FGF21-associated disorders (such as type 1 or type 2 diabetes, obesity or metabolic syndrome) is a pathology associated with the disorders mentioned above. It is an amount that induces, enhances, or otherwise induces an improvement in adverse symptoms, disease progression, physiological state, or resistance to succumbing to the disorder. For the purposes of the present invention, "subject" or "patient" is preferably a human, but is an animal, more specifically a companion animal (e.g. dog, cat, etc.), a livestock (e.g. cow, sheep, pig, horse Etc.) and experimental animals (eg rats, mice, guinea pigs, etc.).

As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" means one or more formulation materials suitable for achieving or enhancing the delivery of the fusion protein of the invention.

The term “antigen” refers to a molecule or portion of a molecule that can be bound by an antibody and further used in an animal to produce an antibody that can bind to the epitope of that antigen. The antigen may have one or more epitopes.

The term "natural Fc" means a molecule or sequence comprising the sequence of a non-antigen-binding fragment produced from digestion of the whole antibody or by other means, regardless of monomeric or multimeric form, and may contain a hinge region. have. The original immunoglobulin source of native Fc is preferably of human origin and may be any immunoglobulin, although IgG1 and IgG2 are preferred. Natural Fc molecules consist of monomeric polypeptides that can be linked in dimeric or multimeric form by covalent (ie disulfide bond) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (eg IgG, IgA and IgE) or subclass (eg IgG1, IgG2, IgG3, IgA1 and IgGA2). to be. One example of a native Fc is a disulfide-binding dimer resulting from papain digestion of IgG (see Elllison et al., 1982, Nucleic Acids Res. 10: 4071-9). The term "natural Fc" as used herein is a generic term for monomer, dimer and multimeric forms.

The term “Fc variant” means a molecule or sequence that is modified from native Fc but still contains a binding site for FcRn (neonatal Fc receptor), which is a salvage receptor. Exemplary Fc variants, as well as interactions with salvage receptors, are described in International Publication Nos. WO 97/34631 and WO 96/32478, which are incorporated herein by reference. Thus, the term “Fc variant” may include molecules or sequences humanized from non-human native Fc. In addition, native Fc includes regions that can be removed because they provide structural features or biological activity that are not required for the fusion molecules of the fusion proteins of the invention. Thus, the term “Fc variant” refers to (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity when expressed in selected host cells, (4) glycosylation, (5 ) One or more natural Fc sites or residues that affect, or (6) bind to, Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC) It includes molecules or sequences that are lacking or that have one or more Fc sites or residues modified. Fc variants are described in more detail below.

The term “Fc domain” includes native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from the whole antibody or produced by other means. In some embodiments of the invention, the Fc domain can be fused to an FGF21 or FGF21 mutant (including a truncated form of the FGF21 or FGF21 mutant), eg, via a covalent bond between the Fc domain and the FGF21 sequence. Such fusion proteins can form multimers through the association of Fc domains, both of which are aspects of the invention.

As used herein, “modified Fc fragment” shall mean an Fc fragment of an antibody comprising a modified sequence. The Fc fragment is part of an antibody comprising CH2, CH3 and part of the hinge region. Modified Fc fragments can be derived, for example, from IgG1, IgG2, IgG3 or IgG4. FcLALA is a modified Fc fragment with LALA mutations (L234A, L235A), which causes ADCC with reduced efficiency and binds to human complement and weakly activates it. Hessell et al. 2007 Nature 449: 101-104. Further modifications to the Fc fragment are described, for example, in US Pat. No. 7,217,798.

The term “heterologous” means that these domains are not found naturally in association with the constant region of the antibody. In particular, this heterologous binding domain does not have the typical structure of an antibody variable domain consisting of four framework regions FR1, FR2, FR3 and FR4 and three complementarity determining regions (CDRs) between them. Thus each arm of the fuzobody comprises a first single chain polypeptide comprising a first binding domain covalently linked at the N-terminal portion of the constant C _H 1 heavy chain region of the antibody, and the N-terminal portion of the constant C _L light chain of the antibody. A second single chain polypeptide comprising a second binding domain covalently linked at. Covalent linkages can be direct, for example via peptidic linkages, or indirect through linkers, eg, peptidic linkers. The two heterodimers of the fuzobody are covalently linked, for example by one or more disulfide bridges in their hinge region, similar to the antibody structure. Examples of molecules having a fuzobody structure, in particular the fuzobody comprising the ligand binding region of the heterodimeric receptor, have been described in the art (see, eg, International Patent Publications WO01 / 46261 and WO11 / 076781).

The term "polyethylene glycol" or "PEG" means a polyalkylene glycol compound or derivative thereof, with or without derivatization with a coupling agent or coupling or activation moiety.

The term “FGF21-associated disorder” and similar terms used herein refers to obesity, type 1 and type 2 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), Insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy , Gastric palsy, disorders associated with severe inactivation mutations in the insulin receptor, and other metabolic disorders.

The term “disorders associated with severe inactivation mutations at insulin receptors” and the term used similarly herein refer to insulin receptors (or, but not always) that show severe (but not always) obesity that is common in type 2 diabetes (or State in a subject suffering from a mutation in a possible protein directly downstream therefrom). In many respects, subjects suffering from these conditions exhibit hybrid symptoms of type 1 diabetes and type 2 diabetes. Subjects suffering from this are approximate severity, including type A insulin resistance, type C insulin resistance (AKA HAIR-AN syndrome), Rapson-Mendenhall syndrome and finally Donohue syndrome or leprosy. It is divided into several categories of increases. These disorders are associated with very high endogenous insulin levels and very often with hyperglycemia. Subjects who suffer from this also exhibit a variety of clinical features associated with "insulin toxicity", including oleodrogenosis, polycystic ovary syndrome (PCOS), hirsutism and melanoma cellulitis (excessive growth and pigmentation) in the folds of the skin.

"Type 2 diabetes" is a condition characterized by excess glucose production despite the availability of insulin, and circulating glucose levels remain excessively high as a result of inadequate glucose clearance.

"Type 1 diabetes" is a condition characterized by high blood glucose levels caused by a complete lack of insulin. This occurs when the body's immune system attacks and destroys insulin-producing beta cells in the pancreas. As a result, the pancreas produces little or no insulin.

"Glucose intolerance" or glucose tolerance disorder (IGT) is a prediabetic state of dysglycemia associated with an increased risk of cardiovascular pathology. A prediabetic condition prevents efficient migration of glucose into cells and use as an efficient fuel source in a subject, resulting in elevated blood glucose levels and some insulin resistance.

"Hyperglycemia" is defined as excess sugar (glucose) in the blood.

Also called hypoglycemia, "hypoglycemia" occurs when blood glucose levels drop too low to provide enough energy for the body's activity.

"Hyperinsulinemia" is defined as insulin in the blood at a higher level than normal.

"Insulin resistance" is defined as a condition in which normal amounts of insulin produce subnormal biological responses.

"Obesity" can be defined as a weight greater than 20% higher than the ideal weight of a given population for human subjects (R. H. Williams, Textbook of Endocrinology, 1974, p. 904-916).

"Diabetic complications" are problems of other physical functions, such as kidneys, nerves (neuropathy), feet (foot ulcers and poor circulation) and eyes (eg retinopathy) caused by high blood glucose levels. In addition, diabetes increases the risk of heart disease and bone and joint disorders. Other long-term complications of diabetes include skin problems, digestive problems, sexual dysfunction and tooth and gum problems.

"Metabolism syndrome" can be defined as a set of three or more of the following signs: abdominal fat-waist more than 40-inch in most men; Hyperglycemia—at least 110 milligrams / deciliter (mg / dl) after fasting; High triglycerides-not less than 150 mg / dL in the bloodstream; Low HDL-less than 40 mg / dl; And blood pressure of at least 130/85 mmHg.

"Panitis" is inflammation of the pancreas.

"Dyslipidemia" is a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency. Dyslipidemia can be manifested by an increase in total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations in the blood, and a decrease in high-density lipoprotein (HDL) cholesterol concentrations.

"Non-alcoholic fatty liver disease (NAFLD)" is a liver disease that is not associated with alcohol consumption, characterized by fatty changes in hepatocytes.

"Nonalcoholic steatohepatitis (NASH)" is a liver disease that is not associated with alcohol consumption, characterized by fat changes in hepatocytes accompanied by intrahepatic inflammation and fibrosis.

"High blood pressure" or high blood pressure is a temporary or sustained elevation of systemic arterial blood pressure to a level likely to induce cardiovascular damage or other adverse consequences. Hypertension has been arbitrarily defined as systolic blood pressure above 140 mmHg or diastolic blood pressure above 90 mmHg.

"Cardiovascular disease" is a disease related to the heart or blood vessels.

"Acute myocardial infarction" occurs when there is a disruption of the blood supply to part of the heart. The resulting ischemia and lack of oxygen can lead to damage or death (infarction) of myocardial tissue (myocardium) if left untreated for a sufficient period of time.

"Peripheral arterial disease" occurs when plaque accumulates in an artery that carries blood to the head, organs, and limbs. Over time, plaques can harden and narrow the arteries, which limits the flow of oxygen-rich blood to organs and other parts of the body.

"Atheromasclerosis" is a vascular disease characterized by lipid deposits that are irregularly distributed in the lining of aorta and medium arteries, causing narrowing of the arterial lumen and eventually progressing to fibrosis and calcification. Lesions are usually local and progress slowly and intermittently. Blood flow restriction accounts for most clinical signs that vary with the distribution and severity of the lesion.

"Stroke" is any acute clinical event regarding a disorder of cerebral circulation that lasts longer than 24 hours. Stroke includes irreversible brain damage, types and severity of symptoms depending on the location and extent of brain tissue with impaired circulation.

Also known as congestive heart failure, "heart failure" is a condition in which the heart can no longer pump enough blood to the rest of the body.

Coronary heart disease, also called coronary artery disease, is the narrowing of the small blood vessels that supply blood and oxygen to the heart.

"Kidney disease" or nephropathy is any kidney disease. Diabetic nephropathy is a major cause of morbidity and death in people with type 1 or type 2 diabetes.

"Neuropathy" is any disease that includes the cranial nerve or the peripheral or autonomic nervous system.

"Gastroparalysis" is a weakening of the stomach peristalsis that causes delayed excretion of the intestines.

Critically ill patients encompassed by the present invention generally experience an unstable troublesome condition. This unstable metabolic state is due to changes in substrate metabolism that can lead to relative deficiency of some nutrients. Generally there is increased oxidation of both fat and muscle.

In addition, the critical patient is preferably a patient experiencing systemic inflammatory response syndrome or dyspnea. Reduction in morbidity means a reduction in the likelihood of developing additional diseases, conditions, or symptoms in critically ill patients or a reduction in the severity of additional diseases, conditions, or symptoms. For example, a reduction in morbidity may correspond to a reduction in the incidence of complications associated with bacteremia or sepsis or multiple organ failure.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, "antibody" includes a mixture of two or more such antibodies.

The term "about" as used herein means +/- 20%, more preferably +/- 10%, even more preferably +/- 5% of the value.

The terms “polypeptide” and “protein” are used interchangeably and refer to polymer forms of amino acids of any length, which modify coding and non-coding amino acids, naturally and non-naturally occurring amino acids, chemically or biochemically. Polypeptides having modified or derivatized amino acids, and modified peptide backbones. The term refers to fusion proteins such as, but not limited to, fusion proteins having heterologous amino acid sequences, heterologous and homologous leader sequences and with or without N-terminal methionine residues; Immunologically tagged proteins and the like.

The terms “individual”, “subject”, “host” and “patient” are used interchangeably and mean any subject, especially a human, in which a diagnosis, treatment or therapy is required. Other subjects can include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments, the subject is a human.

As used herein, the term "sample" refers to a biological material from a patient. Samples assayed by the present invention are not limited to any particular type. Samples include, by way of non-limiting example, single cells, multicells, tissues, tumors, biological fluids, biological molecules, or supernatants or extracts of any of the above. Examples include tissue removed for biopsy, tissue removed during ablation, blood, urine, lymphoid tissue, lymph, cerebrospinal fluid, mucus and stool samples. The sample used will vary depending on the assay format, detection method and the nature of the tumor, tissue, cell or extract being assayed. Methods of making samples are well known in the art and can be readily modified to obtain a sample suitable for the method used.

The term “biological molecule” as used herein includes, but is not limited to, polypeptides, nucleic acids, and saccharides.

The term "modulating" as used herein refers to a change in the quality or quantity of a gene, protein, or any molecule on the inside, outside or surface of a cell. The change can be an increase or decrease in the expression or level of the molecule. The term “regulate” also lowers blood glucose, insulin, triglycerides or cholesterol levels without limitation; Reduce hepatic lipid or hepatic triglyceride levels; Reduce weight; Changes in the quality or amount of biological function / activity, including the ability to improve glucose tolerance, energy expenditure, or insulin sensitivity.

As used herein, the term "modulator" refers to a composition that modulates one or more physiological or biochemical events associated with metabolic conditions such as FGF21-associated disorders such as type 1 or type 2 diabetes or obesity. The event lowers blood glucose, insulin, triglycerides or cholesterol levels; Reduce hepatic lipid or hepatic triglyceride levels; Reduce weight; Including but not limited to the ability to improve glucose tolerance, energy expenditure, or insulin sensitivity.

A "gene product" is a biopolymer product expressed or produced by a gene. Gene products may be, for example, non-spliced RNA, mRNA, splice variant mRNAs, polypeptides, post-translationally modified polypeptides, splice variant polypeptides, and the like. The term also includes biopolymer products (ie cDNA of RNA) prepared using RNA gene products as templates. Gene products may be produced enzymatically, recombinantly, chemically, or in cells in which the gene is native. In some embodiments, if the gene product is proteinic, it exhibits biological activity. In some embodiments, if the gene product is a nucleic acid, it may be translated into a proteinaceous gene product that exhibits biological activity.

As used herein, “modulation of FGF21 activity” refers to, for example, the interaction of an agent with FGF21 polynucleotides or polypeptides, inhibition of FGF21 transcription and / or translation (eg, antisense with FGF21 gene or FGF21 transcript). Or increases or decreases in each of the FGF21 activity, which may be the result of siRNA interactions, through the regulation of transcription factors that facilitate FGF21 expression. For example, modulation of biological activity means increasing or decreasing biological activity. FGF21 activity can be assessed by means including, without limitation, assays of blood glucose, insulin, triglyceride or cholesterol levels, assessment of FGF21 polypeptide levels, or assessment of FGF21 transcription levels in a subject. Comparison of FGF21 activity can also be achieved, for example, by measuring the level of FGF21 downstream biomarkers, and by measuring the increase in FGF21 signaling. FGF21 activity is determined by cell signaling; Kinase activity; Glucose uptake into adipocytes; Fluctuations in blood insulin, triglycerides, or cholesterol levels; Changes in liver lipid or liver triglyceride levels; Interaction between FGF21 and FGF21 receptors; Or by measuring phosphorylation of the FGF21 receptor. In some embodiments, phosphorylation of the FGF21 receptor may be tyrosine phosphorylation. In some embodiments, modulation of FGF21 activity can result in regulation of FGF21-related phenotypes.

Comparison of FGF21 activity can also be achieved, for example, by measuring the level of FGF21 downstream biomarkers, and by measuring the increase in FGF21 signaling. FGF21 activity is determined by cell signaling; Kinase activity; Glucose uptake into adipocytes; Fluctuations in blood insulin, triglycerides, or cholesterol levels; Changes in liver lipid or liver triglyceride levels; Interaction between FGF21 and the receptor (FGFR-1c, FGFR-2c, or FGFR-3c); Or by measuring phosphorylation of the FGF21 receptor. In some embodiments, phosphorylation of the FGF21 receptor may be tyrosine phosphorylation. In some embodiments, modulation of FGF21 activity can result in regulation of FGF21-related phenotypes.

"FGF21 downstream biomarker" as used herein is a gene or gene product, or a measurable indication of a gene or gene product. In some embodiments, the gene or activity that is a downstream marker of FGF21 exhibits altered expression levels or appears in vascular tissue. In some embodiments, the activity of the downstream marker is altered in the presence of a FGF21 modulator. In some embodiments, the downstream markers indicate altered expression levels when FGF21 is disrupted by the FGF21 modulator of the invention. FGF21 downstream markers include, without limitation, glucose or 2-deoxy-glucose uptake, pERK and other phosphorylated or acetylated protein or NAD levels.

The term "up-regulate" as used herein means an activity or an increase, activation or stimulation of an amount. For example, in the context of the present invention, FGF21 modulators can increase the activity of the FGF21 receptor. In an embodiment, one or more FGFR-1c, FGFR-2c or FGFR-3c can be up-regulated in response to FGF21 modulator. Up-regulation also lowers, for example, blood glucose, insulin, triglycerides or cholesterol levels; Reduce hepatic lipid or hepatic triglyceride levels; Reduce weight; Improve glucose tolerance, energy expenditure, or insulin sensitivity; Or causes phosphorylation of the FGF21 receptor; Or FGF21-related activity such as the ability to increase FGF21 downstream markers. The FGFR21 receptor may be one or more of FGFR-1c, FGFR-2c or FGFR-3c. Up-regulation can be at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 400% or at least 500% relative to the control.

The term "N-terminus" as used herein refers to at least the first 20 amino acids of a protein.

As used herein, the terms "N-terminal domain" and "N-terminal region" are used interchangeably and refer to a fragment of a protein starting at the first amino acid of the protein and ending at any amino acid in the N-terminal half of the protein. it means. For example, the N-terminal domain of FGF21 is from amino acid 1 of SEQ ID NO: 1 to approximately any amino acid between amino acids 10 and 105 of SEQ ID NO: 1.

The term "C-terminus" as used herein means at least the last 20 amino acids of the protein.

As used herein, the terms “C-terminal domain” and “C-terminal region” are used interchangeably and refer to a fragment of a protein starting at any amino acid in the C-terminal half of the protein and ending at the last amino acid of the protein. it means. For example, the C-terminal domain of FGF21 begins at any amino acid from amino acid 105 of SEQ ID NO: 1 to approximately amino acid 200 and ends at amino acid 209 of SEQ ID NO: 1.

The term "domain" as used herein refers to the structural part of a biomolecule that contributes to the known or putative function of the biomolecule. A domain may extend simultaneously with a region or portion thereof, and may include a portion of a biomolecule that is distinct from a particular region in addition to all or a portion of the region.

As used herein, the term “signal domain” (also called “signal sequence” or “signal peptide”) refers to a peptide present in the continuous stretch of amino acid sequence in the N-terminal region of a precursor protein (often a membrane binding or secreting protein). Refers to a domain and is involved in posttranslational protein transport. In many cases, the signal domain is removed from the full-length protein by special signal peptidase after the classification process is completed. Each signal domain specifies a specific destination within the cell for the precursor protein. The signal domain of FGF21 is amino acids 1-28 of SEQ ID NO: 1.

As used herein, the term “receptor binding domain” refers to any portion or region of a protein that contacts a membrane binding receptor protein to cause a cellular response, such as a signaling event.

As used herein, the term “ligand binding domain” means any portion or region of the fusion protein of the invention that retains one or more qualitative binding activities of the corresponding native sequence.

The term "region" refers to the physically contiguous portion of the primary structure of a biomolecule. In the case of a protein, a region is defined by contiguous portions of the amino acid sequence of the protein. In some embodiments a “region” is associated with the function of a biomolecule.

As used herein, the term "fragment" refers to the physically contiguous portion of the primary structure of a biomolecule. In the case of a protein, the portion is defined by contiguous portions of the amino acid sequence of the protein and includes at least 3-5 amino acids, at least 8-10 amino acids, at least 11-15 amino acids, at least 17-24 amino acids, At least 25-30 amino acids, and at least 30-45 amino acids. In the case of an oligonucleotide, said portion is defined by the contiguous portion of the nucleic acid sequence of the oligonucleotide, at least 9-15 nucleotides, at least 18-30 nucleotides, at least 33-45 nucleotides, at least 48-72 Nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides. In some embodiments, the portion of the biomolecule has biological activity. In the context of the present invention, the FGF21 polypeptide fragment does not comprise the entire FGF21 polypeptide sequence set forth in SEQ ID NO: 1.

A “natural sequence” polypeptide is one having the same amino acid sequence as a polypeptide derived from nature. Such native sequence polypeptides can be isolated from nature or can be prepared by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring human polypeptide, a murine polypeptide, or a polypeptide from any other mammalian species.

As used herein, the phrase “homologous nucleotide sequence”, or “homologous amino acid sequence”, or a modification thereof, means a sequence characterized by at least a specified percentage of homology at the nucleotide level or at the amino acid level, and with “sequence identity” Used interchangeably. Homologous nucleotide sequences include sequences encoding isotypes of proteins. Such isotypes can be expressed in different tissues of the same organism, for example as a result of selective splicing of RNA. Alternatively, the isotypes can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences that encode proteins of species other than human, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Homologous amino acid sequences include amino acid sequences that contain conservative amino acid substitutions and in which the polypeptide has the same binding and / or activity. In some embodiments, the nucleotide or amino acid sequence is homologous if it has at least 60% or more, up to 99% identity with the comparative sequence. In some embodiments, a nucleotide or amino acid sequence is homologous if it shares one or more, up to 60 nucleotide / amino acid substitutions, additions or deletions with the comparison sequence. In some embodiments, homologous amino acid sequences have up to 5 or up to 3 conservative amino acid substitutions.

Percent homology or identity is, for example, a gap using default settings, using Smith and Waterman algorithms (Adv. Appl. Math., 1981, 2, 482-489). It can be determined by the program (Wisconsin Sequence Analysis Package for UNIX, Version 8, Genetics Computer Group, Madison University Research Park, Wisconsin). In some embodiments, homology between the probe and the target is about 75% to about 85%. In some embodiments, the nucleic acid has a nucleotide that is at least about 95%, about 97%, about 98%, about 99%, and about 100% homologous to SEQ ID NO: 2 or a portion thereof.

Homology can also be at the polypeptide level. In some embodiments, the component polypeptides of the fusion proteins of the invention may be at least 95% homologous to their full length wild type counterpart or corresponding native sequence, or portion thereof. The degree of identity or percentage of different amino acid sequences with the fusion protein or portion thereof of the present invention is determined by the number of exact matches in the alignment of the two sequences divided by the length of the shortest of either the "sequence sequence" or the "foreign sequence". Is calculated. The result is expressed as percent identity.

As used herein, the term "mixing" refers to the process of combining one or more compounds, cells, molecules, etc. together in the same region. This can be done, for example, in a test tube, petri dish, or any vessel that allows mixing of one or more compounds, cells, or molecules.

As used herein, the term “substantially purified” refers to compounds (eg, at least 60%, at least 75%, and at least 90% free of other components that are removed from their natural environment and naturally associated therewith (eg, Polynucleotide or polypeptide or antibody).

The term "pharmaceutically acceptable carrier" means a carrier for the administration of a therapeutic agent such as an antibody or polypeptide, gene, and other therapeutic agent. The term itself refers to any pharmaceutical carrier that can be administered without excessive toxicity, without inducing the production of antibodies that are harmful to the individual to whom the composition has been administered. Suitable carriers can be large and slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates and inert viral particles. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids such as water, saline, glycerol and ethanol. Auxiliary materials such as wetting or emulsifying agents, pH buffering materials and the like may also be present in such vehicles.

Enhancement of Physical Stability of Fusion Proteins of the Invention

Naturally occurring disulfide bonds, as provided by cysteine residues, generally increase the thermodynamic stability of the protein. Successful examples of increased thermodynamic stability as measured at an increase in melting temperature are the enzyme T4 lysozyme (Matsumura et al., PNAS 86: 6562-6566 (1989)) and varase (Johnson et al., J). Mol. Biol. 268: 198-208 (1997)). An aspect of the present invention is the enhancement of physical stability of FGF21 in the presence of a preservative, which is achieved by the presence of disulfide bonds in variants that limit the flexibility of wild type FGF21 to limit the access of the preservative to the hydrophobic core of the protein.

The second aspect of the invention thus has improved pharmaceutical stability induced by the incorporation of further disulfide bonds, for example, by incorporation or substitution of a cysteine residue into a wild type FGF21 protein or a polypeptide and a protein variant of the invention. Variants of human FGF21, or biologically active peptides thereof. Those skilled in the art will appreciate that natural cysteine, cysteine 103 and cysteine 121 can be used as loci for introducing novel disulfide bonds that can confer improved properties other than the proposed embodiments described herein.

These include fusion proteins incorporating cysteine substitution for two or more of the following in wild type FGF-21: glutamine 46, arginine 47, tyrosine 48, leucine 49, tyrosine 50, threonine 51, aspartate 52, Aspartate 53, alanine 54, glutamine 55, glutamine 56, threonine 57, glutamate 58, alanine 59, histidine 60, leucine 61, glutamate 62, isoleucine 63, valine 69, glycine 70, glycine 71, alanine 72, alanine 73, Leucine 144, Histidine 145, Leucine 146, Proline 147, Glycine 148, Asparagine 149, Lysine 150, Serine 151, Proline 152, Histidine 153, Arginine 154, Aspartate 155, Proline 156, Alanine 157, Proline 158, Arginine 159, Glycine 160, proline 161, alanine 162, arginine 163, phenylalanine 164, wherein the numbering of amino acids is based on the full-length 209 amino acid hFGF21 sequence (SEQ ID NO: 1).

In addition, the fusion proteins of the invention may include variants of wild-type human FGF21, or biologically active peptides thereof, enhanced by engineered disulfide bonds other than those naturally occurring in Cys103-Cys121, such as:

Another aspect of the invention is any charged and / or at any amino acid position shown in the first embodiment of the invention in combination with the substitution of cysteine at two or more amino acid positions shown in the second embodiment of the invention. Provided are fusion proteins comprising variants of wild-type human FGF21, or biologically active peptides thereof, that include substitution of polar but uncharged amino acids.

Improvement of the fusion protein of the invention to wild type protein comparisons and variants thereof

It is well known in the art that significant problems in the development of protein pharmaceuticals deal with the physical and chemical instability of proteins. This is even more evident when protein pharmaceutical formulations are intended as multi-use injectable formulations that require stable concentration and preservation solutions while maintaining an advantageous bioactivity profile. Biophysical characterization of wild-type FGF21 in the literature indicates accelerated association and aggregation (ie, poor physical stability and biopharmaceutical properties) when concentrated protein solutions (greater than 5 mg / ml) are exposed to stress conditions such as high temperature or low pH. Induction was established. Exposure to the pharmaceutical preservative (eg m-cresol) of the concentrated protein solution of FGF21 also had a negative effect on physical stability.

Accordingly, an embodiment of the present invention is to enhance the physical stability of a concentrated solution while maintaining chemical stability and biological potency under both physiological and preserved formulation conditions. Since temperature and ionic strength have a significant impact on physical stability at a given protein concentration, it is contemplated that association and aggregation may be the result of hydrophobic interactions. In most cases, non-conserved, presumed surface exposed amino acid residues were targeted. The local environment of these residues was analyzed and residues not considered structurally important were selected for mutagenesis. One way to initiate certain changes was to further reduce the pi of the protein by introducing glutamic acid residues (“glutamic acid scan”). It is hypothesized that the introduction of charged substituents will inhibit hydrophobic-mediated aggregation through charge-charge repulsion, potentially improving preservative compatibility. Those skilled in the art will also recognize that, with a sufficient degree of mutagenesis, pI can shift to the basic pH range by allowing charge-charge repulsion by introduction of positive charges with or without simultaneous reduction of negative charges.

A further problem associated with the therapeutic use of wild type FGF21 as a biotherapeutic agent is that its half-life, for example, is very short in vivo (about 0.5 and 2 hours in mice and primates, respectively). Thus there is a need to develop more effective subsequent compounds with higher potency or longer half-life. The fusion proteins of the invention have been developed in such a way as to achieve the desired effect of FGF21 treatment with higher potency and half-life-extended formulations.

Further fusion proteins of the invention as described herein have a much shorter half-life of wild type FGF21 and a 17-hour half-life of the fusion protein Fc-L (15) -FGF21 (L98R, P171G, A180E) in PCT Publication WO10 / 129600. In comparison, mice have half-lives longer than two weeks. The fusion proteins of the invention also demonstrate improved half-life and pharmacokinetic properties compared to PEGylated V76 as described herein and in US Patent Application 61 / 415,476, filed November 19, 2010.

In addition, 1 mpk of the Fc-FGF21 fusion protein of the invention is more effective than 5 mpk of V76 for the reduction of glucose, insulin, weight and liver lipids. In a 12-day treatment study of ob / ob mice, the fusion protein shows the percent change from the following vehicles (all fusions are administered at 1.0 mg / kg and V76 is administered at 5.0 mg / kg):

% Change in total glucose (AUC) from vehicle: V76 is -42%; V101 is -53%, V103 is -46%, and V188 is -42%;

% Change in total plasma insulin from vehicle: V76 is -46%; V101 is -82%, V103 is -69%, and V188 is -59%;

% Total weight change from vehicle: V76 is -7%; V101 is -12%, V103 is -12%, and V188 is -11%;

% Change in total liver lipids from vehicle: V76 is -30%; V101 is -44%, V103 is -50% and V188 is -51%.

Similarly, in vitro assays show the same five-fold or higher potency of the fusion proteins of the invention on V76:

In pERK in human adipocyte assay (mean EC50 ± SEM), V76 is 21 ± 2 nM (n = 3); V101 is 1.0 ± 0.1 nM (n = 3), V103 is 1.3 ± 0.2 nM (n = 3) and V188 is 1.4 ± 0.4 nM (n = 3);

At pERK in HEK293 by human β-cloto assay (mean EC50 ± SEM), V76 is 13 ± 4 nM (n = 5), V101 is 0.60 ± 0.06 nM (n = 5), and V103 is 0.9 ± 0.3 nM (n = 5) and V188 is 0.4 ± 0.1 nM (n = 3);

In glucose uptake in mouse adipocyte assay (mean EC50 ± SEM), V76 is 5 ± 1 nM (n = 3), V101 is 0.60 ± 0.06 nM (n = 3) and V103 is 0.60 ± 0.07 nM (n = 3) and V188 is 0.48 ± 0.14 nM (n = 3).

Embodiments of the invention relate to physical and chemical stability under both physiological and conserved pharmaceutical formulation conditions, but maintaining the biological potency of the fusion proteins of the invention is also an important consideration factor when compared to, for example, wild type FGF21. . Thus, the biological potency of the proteins of the invention is defined by the ability of the proteins to affect glucose uptake and / or lowering of plasma glucose levels as shown in the Examples herein.

Proteins, polypeptides and / or peptides of the invention administered in accordance with the invention may be produced and / or isolated by any means known in the art. The most preferred method for generating variants is via recombinant DNA methodology and is well known to those skilled in the art. Such methods are described in Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), incorporated herein by reference.

In addition, preferred embodiments include biologically active peptides derived from the variants described herein. Such peptides will contain one or more substitutions described, and the variant will retain biological activity. Peptides can be produced by any and all means known to those skilled in the art, examples of which include, but are not limited to, enzymatic digestion, chemical synthesis, or recombinant DNA methodology.

It is established in the art that peptide fragments of certain fibroblast growth factors have biological activity. See, eg, aird et al., Proc. Natl. Acad. Sci (USA) 85: 2324-2328 (1988), and J. Cell. Phys. Suppl. 5: 101-106 (1987). Thus, the selection of fragments or peptides of the variants is based on criteria known in the art. Dipeptidyl peptidase IV (DPP-IV or DPP-4), for example, is known as serine-type proteases associated with inactivation of neuropeptides, endocrine peptides and cytokines (Damme et al. Chem. Immunol. 72 : 42-56, (1999)]. The N-terminus of FGF21 (HisProIlePro) is potentially a substrate of DPP-IV and thus contains two dipeptides capable of producing fragments of FGF21 truncated at four N-terminus. Unexpectedly, this fragment of wild-type FGF21 has been shown to retain biological activity, so a protein of the invention wherein up to four amino acids are truncated at the N-terminus are embodiments of the invention.

The invention also encompasses polynucleotides encoding the above-described variants, which may be in the form of RNA or in the form of DNA, wherein the DNA comprises cDNA, genomic DNA and synthetic DNA. DNA can be double-stranded or single-stranded. Coding sequences encoding proteins of the invention may vary as a result of redundancy or degeneracy of the genetic code.

Polynucleotides encoding the fusion protein of the invention may include the following: coding sequence alone for a variant; Coding sequences for variants and additional coding sequences, such as functional polypeptides, or leader or secretory sequences or pro-protein sequences; Coding sequences and non-coding sequences for the variant, such as the 5 'and / or 3' intron or non-coding sequence of the coding sequence for the variant. Thus, the term “polynucleotide encoding a variant” includes polynucleotides that may include polynucleotides that include additional coding and / or non-coding sequences as well as coding sequences for the variants.

The invention further relates to variants of the described polynucleotides encoding fragments, analogs and derivatives of the polypeptides containing the indicated substitutions. Variants of the polynucleotides may be naturally occurring allelic variants, non-naturally occurring variants of the human FGF21 sequence, or truncated variants described above. Accordingly, the present invention also encompasses polynucleotides encoding the variants described above, as well as variants of such polynucleotides in which the variants encode fragments, derivatives or analogs of the disclosed variants. Such nucleotide variants include deletion variants, substitution variants, truncated variants, and addition or insertion variants, so long as one or more of the indicated amino acid substitutions of the first or second embodiment are present.

Polynucleotides of the invention will be expressed in the host after the sequence is operably linked (ie located to ensure function) to the expression control sequence. These expression vectors are typically replicable in the host organism as episomal or as an integral part of the host chromosomal DNA. Typically, expression vectors will contain selection markers such as tetracycline, neomycin and dihydrofolate reductase to allow detection of these cells transformed with the DNA sequence of interest. FGF21 variants may be expressed in mammalian cells, insects, yeasts, bacteria or other cells under the control of appropriate promoters. Cell-free translation systems may be used to produce such proteins using RNA derived from the DNA constructs of the invention.

this. E. coli are particularly useful prokaryotic hosts for cloning the polynucleotides of the present invention. Other microbial hosts suitable for use are Bacillus subtilus, Salmonella typhimurium, and various species of Serratia, Pseudomonas, Streptococcus, and Staphylococcus. Includes Staphylococcus, but others may be used as desired. In these prokaryotic hosts, expression vectors may also be prepared that will contain expression control sequences (eg, origin of replication) that are compatible with the host cell. There may also be any of a number of well known promoters, such as the lactose promoter system, tryptophan (Trp) promoter system, beta-lactamase promoter system, or promoter system from phage lambda or T7. Promoters will typically have ribosomal binding site sequences, etc., to control expression, initiate and complete transcription and translation, optionally with operator sequences.

One of ordinary skill in the art of protein expression is E. For expression in E. coli, it will be appreciated that methionine or methionine-arginine sequences may be introduced at the N-terminus of the mature sequence (SEQ ID NO: 3) and are contemplated within the context of the present invention. Thus, unless otherwise indicated, this. Proteins of the invention expressed in E. coli have a methionine sequence introduced at the N-terminus.

Other microorganisms such as yeast or fungi can also be used for expression. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia angusta are examples of preferred yeast hosts and are intended. Promoters comprising an expression control sequence as such, such as 3-phosphoglycerate kinase or other enzymes of interest, and suitable vectors with origins of replication, termination sequences, and the like. Aspergillus niger, Trichoderma reesei; And Schizophyllum commune are examples of fungal hosts, but others may be used as desired.

Mammalian tissue cell culture can also be used to express and produce the polypeptides of the invention. Eukaryotic cells are actually preferred because many suitable host cell lines have been developed in the art that can secrete intact variants, including CHO cell lines, various COS cell lines, NSO cells, and Syrian Hamster ovary. Cell lines, HeLa cells, or human embryonic kidney cell lines (ie, HEK293, HEK293EBNA).

Expression vectors for these cells can include expression control sequences such as origin of replication, promoters, enhancers, and necessary processing information sites such as ribosomal binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. . Preferred expression control sequences are promoters derived from SV40, adenovirus, bovine papilloma virus, cytomegalovirus, Raus sarcoma virus, and the like. Preferred polyadenylation sites include sequences derived from SV40 and bovine growth hormone.

Vectors containing polynucleotide sequences of interest (eg, fusion proteins and expression control sequences of the invention) can be delivered into host cells by well known methods that vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular hosts.

Various protein purification methods can be used and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182: 83-9 (1990) and Scopes, Protein Purification: Principles and Practice, Springer Verlag, NY (1982). The purification step (s) selected will depend, for example, on the nature of the production method used in the fusion proteins of the invention.

Proteins, polypeptides and / or peptides of the invention, such as dual active fusion proteins of the invention, are excellent in view of the clinical condition of the patient, the site of delivery of the protein composition, the method of administration, the dosing regimen, and other factors known to the physician. It must be formulated and administered in a manner consistent with medical practice. Thus, the "therapeutically effective amount" of the fusion protein of the invention for the purposes herein is determined by these considerations.

Pharmaceutical compositions of the proteins of the invention may be administered by any means which achieve generally intended aims for the treatment of type 1 and type 2 diabetes, obesity, metabolic syndrome or critically ill. Non-limiting acceptable means of administration are, for example, by inhalation or suppositories or by washing into mucosal tissues, such as vaginal, rectal, urethral, buccal and sublingual tissues, orally, nasal, topically, intranasally, Administration intraperitoneally, parenterally, intravenously, intramuscularly, intrasternally, by intra-articular injection, intralymphatically, epilepsy, intraarterally, subcutaneously, intravitreal, transepithelial, and transdermally. In some embodiments, the pharmaceutical composition is administered by washing, orally, or intraarterially. Other suitable introduction methods may also include rechargeable or biodegradable devices and slow or sustained release polymeric devices. Pharmaceutical compositions of the invention may also be administered as part of a combination therapy with other known metabolic agents.

The dosage administered will depend on the age, health and weight of the recipient, the type of concurrent treatment, the frequency of treatment, and the nature of the desired effect if present. Compositions within the scope of the present invention include all compositions in which the FGF21 variant is present in an amount effective to achieve the desired medical effect for the treatment of type 1 or type 2 diabetes, obesity or metabolic syndrome. While individual requirements may vary from patient to patient, it is within the ability of clinicians of ordinary skill to determine the optimal range for an effective amount of all components.

Proteins of the invention can be formulated according to known methods of preparing pharmaceutically useful compositions. The desired formulation may be a stable lyophilized product which is reconstituted with an appropriate diluent or a high purity aqueous solution containing any pharmaceutically acceptable carrier, preservative, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition (1980)). . Proteins of the invention can be combined with pharmaceutically acceptable buffers, and pH adjusted to provide acceptable stability, and pH acceptable for administration.

For parenteral administration, in one embodiment, the fusion protein of the invention is generally one or more proteins of the desired purity and is non-toxic to the recipient at the pharmaceutically acceptable carrier, i. It is formulated by mixing in an injectable unit dosage form (solution, suspension or emulsion) with those compatible with. Preferably, one or more pharmaceutically acceptable anti-microbial agents may be added. Phenol, m-cresol and benzyl alcohol are preferred pharmaceutically acceptable anti-microbial agents.

Optionally, one or more pharmaceutically acceptable salts may be added to adjust the ionic strength or tonicity. One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin, sodium chloride and mannitol are examples of isotonicity adjusting excipients.

One skilled in the art can readily optimize the pharmaceutically effective dosages and dosing regimens for therapeutic compositions comprising the proteins of the invention, as determined by good medical practice and the clinical condition of the individual patient. Typical dosage ranges for proteins of the invention will be from about 0.01 mg / day to about 1000 mg / day (or from about 0.05 mg / week to about 5000 mg / week administered once per week) in adults. Preferably, the dosage range is from about 0.1 mg / day to about 100 mg / day (or from about 0.5 mg / week to about 500 mg / week administered once per week), more preferably from about 1.0 mg / day to about 10 mg / day (or about 5 mg / week to about 50 mg / week administered once per week). Most preferably, the dosage is about 1-5 mg / day (or about 5 mg / week to about 25 mg / week administered once per week). Appropriate doses of the FGF21 variant administered will lead to lower blood glucose levels and increased energy consumption by faster and more efficient use of glucose and are therefore useful for treating type 1 and type 2 diabetes, obesity and metabolic syndrome.

In addition, since hyperglycemia and insulin resistance are common in critically ill patients given nutritional support, some ICUs administer insulin to treat excessive hyperglycemia in critically ill patients. Indeed, recent studies have documented that the use of exogenous insulin to maintain blood glucose levels below 110 mg / dl has reduced morbidity and mortality in critically ill patients in the surgical intensive care unit, regardless of the presence of a history of diabetes. Van den Berghe, et al. N Engl J Med., 345 (19): 1359, (2001). Thus, the proteins of the present invention are uniquely suited to aid in the recovery of metabolic stability in metabolic unstable critical patients. Proteins of the invention, such as those containing a variant of FGF21, are unique in that they stimulate glucose uptake and enhance insulin sensitivity but do not induce hypoglycemia.

In another aspect of the invention, obesity, type 1 and type 2 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose Proteins of the invention are contemplated for use as a medicament for the treatment of intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, conditions associated with severe inactivation mutations in the insulin receptor, and other metabolic disorders.

Site-specific FGF21 Mutant

In some embodiments, fusion proteins of the invention comprise additional FGF21 mutants or FGF21 analogs with non-natural amino acids.

In some embodiments, a fusion protein of the invention comprises an FGF21 agonist having one or more of the following additional modifications of wild type FGF21:

(i) dimerization, such as the formation of disulfide at R154C or the introduction of cysteine at another site, or dimerization via fused Fc domains, or dimerization via crosslinking agents such as difunctional PEG Further disulfides, unnatural amino acids, or modifications;

(ii) a fragment of FGF21;

(iii) a protein selected to have FGF21 activity (binding to beta-cloto and binding and activation of FGFR); And

(iv) FGF21 mimicking antibodies (various formats such as Fab, unibody, svFc, etc.).

In some embodiments, the fusion proteins of the invention comprise one or more of the following linkers: simple amide bonds, short peptides (especially Ser / Gly repeats), further residues from the FGF21 translated sequence, or more, to the entire protein. Large linkers (eg Fc domains, HSA-binding helix bundles, HSA, etc.). The two moieties may also be linked by other chemical means, such as through non-natural amino acids or standard chemical linkers (maleimide-Cys, NHS-Lys, click, etc.).

Other embodiments of the invention include, but are not limited to, the following attachments for half-life extension: HSA-binding lipids or small molecules or micelles for monomeric or dimeric versions of the fusion.

In certain embodiments of the invention, other attachments may be made to the proteins, polypeptides and / or peptides of the invention to achieve half-life extension and other improved biological properties. These include attaching PEG-cholesterol conjugates (including micelles and liposomes) to proteins, polypeptides and / or peptides of the invention, and / or attaching sugars (glycosylates) to proteins, polypeptides and / or peptides of the invention. can do. In another embodiment, similar techniques are employed to add, for example, conjugates of polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligand, or carbohydrate shielding to proteins, polypeptides, and / or peptides. Used.

HESylation techniques couple branched hydroxyethylstarch (HES) chains (60 kDa or 100 kDa, highly branched amylopectin fragments from corn starch) to proteins, polypeptides and / or peptides, for example, via reductive alkylation. Let's do it. Polysialication conjugates the protein, polypeptide and / or peptide of interest with a polysialic acid (PSA) polymer in a similar manner to PEGylation. PSA polymers are negatively charged non-immunogenic polymers naturally occurring in the body and are available in molecular weights of 10-50 kD.

In another embodiment of the invention, other attachments or modifications may be made to the proteins, polypeptides and / or peptides of the invention to achieve half-life extension and other improved biological properties. These include the generation of recombinant PEG (rPEG) groups and their attachment to the proteins, polypeptides and / or peptides of the invention. The rPEG technology, as developed by the company Amunix, Inc., is based on protein sequences with PEG-like properties genetically fused to biopharmaceuticals, thus avoiding further chemical conjugation steps. rPEG is an extended half-life exenatide construct containing a long unstructured tail of hydrophilic amino acids, which allows both an increase in serum half-life of the protein or peptide and a decrease in its rate of absorption, thereby significantly reducing the peak-to-bottom ratio. Can be. rPEG has an increased hydrodynamic radius, exhibits an apparent molecular weight that is about 15 times its actual molecular weight, and mimics the PEGylation scheme to achieve long serum half-life.

Truncated FGF21 Polypeptide

One embodiment of the invention relates to a truncated form of a mature FGF21 polypeptide (SEQ ID NO: 3). This embodiment of the present invention has been accomplished by an effort to identify truncated FGF21 polypeptides that can provide superior activity in some cases, similar to the non-terminated form of mature FGF21 polypeptide.

As used herein, the term “terminated FGF21 polypeptide” means that the amino acid residue has been removed from the amino-terminal (or N-terminal) end of the FGF21 polypeptide, or that the amino acid residue is the carboxyl-terminal (or C-terminal) of the FGF21 polypeptide. Means an FGF21 polypeptide that has been removed from an end or an amino acid residue has been removed from both the amino- and carboxyl-terminal ends of the FGF21 polypeptide. The various terminal sections disclosed herein were prepared as described herein.

The activity of the N-terminally truncated FGF21 polypeptide and the C-terminally truncated FGF21 polypeptide can be assayed using an in vitro phospho-ERK assay. Specific details of in vitro assays that can be used to investigate the activity of truncated FGF21 polypeptides can be found in the Examples.

The activity of the truncated FGF21 polypeptides of the invention can also be assessed in vivo assays such as ob / ob mice. In general, the truncated FGF21 polypeptide may be administered intraperitoneally to a test animal to assess the in vivo activity of the truncated FGF21 polypeptide. After the desired incubation period (eg 1 hour or more), blood samples may be taken and blood glucose levels measured.

a. Truncation at the N-terminus

In some embodiments of the invention, truncation at the N-terminus comprises 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residues from the N-terminal end of the mature FGF21 polypeptide. Truncated FGF21 polypeptides with truncations at the N-terminus of less than 9 amino acid residues retain the ability of a mature FGF21 polypeptide to lower blood glucose in an individual. Thus, in certain embodiments, the present invention provides truncated forms of mature FGF21 polypeptides or FGF21 protein variants with truncations at the N-terminus of 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residues. It includes.

b. Truncation at the C-terminus

In some embodiments of the invention, truncation at the C-terminus is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 from the C-terminus of the mature FGF21 polypeptide. Amino acid residues. Truncated FGF21 polypeptides with truncations at the C-terminus of less than 13 amino acid residues showed at least 50% of the efficacy of wild type FGF21 in an in vitro ELK-luciferase assay (see, Jie et al. FEBS Letts 583: 19-24 (2009)]), indicating that these FGF21 mutants retain the ability of mature FGF21 polypeptides to lower blood glucose in an individual. Thus, in certain embodiments, the present invention provides a mature FGF21 polypeptide having a truncation at the C-terminus of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues or Truncated forms of the FGF21 protein variant.

c. Truncation at the N- and C-terminus

In some embodiments of the invention, the truncated FGF21 polypeptide may have a combination of truncation at the N-terminus and C-terminus. Truncated FGF21 polypeptides having a combination of truncation at the N- and C-terminus share the activity of the corresponding truncated FGF21 polypeptide having only truncation at the N- or C-terminus. That is, truncated FGF21 polypeptides having both truncations at the N-terminus of less than 9 amino acid residues and truncations at the C-terminus of less than 13 amino acid residues are N-terminus of less than 9 amino acid residues. Possesses similar or higher blood glucose-lowering activity than truncated FGF21 polypeptide with truncation in or truncated FGF21 polypeptide with truncation at the C-terminus of less than 13 amino acid residues. Thus, in certain embodiments, the invention provides truncation at the N-terminus of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues and 1, 2, 3, 4, 5, 6, Truncated forms of mature FGF21 polypeptide or FGF21 protein variant, with truncations at the C-terminus of 7, 8, 9, 10, 11, or 12 amino acid residues.

As with all FGF21 variants of the invention, the truncated FGF21 polypeptide may optionally comprise amino-terminal methionine residues that can be introduced by a designated mutation or as a result of the bacterial expression process.

Truncated FGF21 polypeptides of the invention can be prepared as described in the Examples described herein. Those skilled in the art, who are familiar with standard molecular biology techniques, can use that knowledge in conjunction with the disclosure herein to prepare and use the truncated FGF21 polypeptides of the invention. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (eg electroporation, lipofection). See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, incorporated herein by reference for any purpose. Enzymatic reactions and purification techniques can be performed according to manufacturer's details, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature and experimental procedures and techniques used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques for chemical synthesis; Chemical analysis; Pharmaceutical preparation, formulation, and delivery; And for the treatment of patients.

Truncated FGF21 polypeptides of the invention can also be fused to another substance that can impart additional properties to the truncated FGF21 polypeptide. In one embodiment of the invention, the truncated FGF21 polypeptide may be fused to an IgG constant domain or fragment thereof (eg an Fc region), human serum albumin (HSA), or albumin-binding polypeptide. Such fusion can be accomplished using known molecular biological methods and / or instructions provided herein. The benefits of such fusion polypeptides, as well as methods of making such fusion polypeptides, are described in more detail herein.

FGF21 fusion protein

As used herein, the term “FGF21 fusion polypeptide” or “FGF21 fusion protein” refers to one or more amino acid residues (eg, heterologous proteins or peptides) at the N-terminus or C-terminus of any of the FGF21 protein variants described herein. Means a fusion.

FGF21 fusion proteins can be prepared by fusing heterologous sequences, eg, at the N-terminus or C-terminus of the FGF21 protein variant as defined herein. The heterologous sequence as described herein may be an amino acid sequence or a non-amino acid-containing polymer. The heterologous sequence can be fused directly to the FGF21 protein variant or through a linker or adapter molecule. The linker or adapter molecule may comprise one or more amino acid residues (or -mers), for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues (or -mers), preferably 10 to 50 Dog amino acid residues (or -mers), for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 residues (or- Dimers), more preferably 15 to 35 amino acid residues (or -mers). The linker or adapter molecule may also be designed to have a cleavage site for DNA restriction endonucleases or for proteases that allow for separation of the fused moiety.

Heterologous peptides and polypeptides include epitopes that allow detection and / or isolation of FGF21 protein variants; Transmembrane receptor proteins or portions thereof, such as extracellular domains or transmembrane and intracellular domains; Ligands or portions thereof that bind to the transmembrane receptor protein; Enzymes or portions thereof having catalytic activity; Polypeptides or peptides that promote oligomerization, such as leucine zipper domains; Polypeptides or peptides such as immunoglobulin constant regions that increase stability; Functional or non-functional antibodies, or heavy or light chains thereof; And polypeptides having a different activity, such as therapeutic activity, from the FGF21 protein variant of the invention. Also included in the present invention are FGF21 mutants fused to human serum albumin (HSA).

a. Fc fusion

In one embodiment of the invention, the FGF21 protein variant is fused to one or more domains of the Fc region of human IgG. Antibodies comprise two functionally independent parts, a variable domain known as "Fab" that binds to an antigen and a constant domain known as "Fc" associated with effector functions such as complement activation and attack by phagocytes. Fc has a long serum half-life, while Fab has a short lifespan (Capon et al., 1989, Nature 337: 525-31). When linked with therapeutic proteins, the Fc domain can provide longer half-life or incorporate functions such as Fc receptor binding, protein A binding, complement fixation, and possibly even placental delivery (Capon et al., 1989).

Throughout the disclosure, Fc-FGF21 refers to a fusion protein in which the Fc sequence is fused to the N-terminus of FGF21. Similarly, throughout the disclosure, FGF21-Fc means a fusion protein in which the Fc sequence is fused to the C-terminus of FGF21.

Preferred embodiments of the invention are Fc-FGF21 fusion proteins comprising the FGF21 variant as defined herein. Particularly preferred embodiments are Fc-FGF21 fusion proteins comprising modified Fc fragments (eg FcLALA) and FGF21 variants as defined herein.

Fusion proteins can be purified, for example, by use of a Protein A affinity column. Peptides and proteins fused to the Fc region have been found to exhibit substantially longer half-lives than unfused counterparts in vivo. Fusion to the Fc region also allows for dimerization / multimerization of the fusion polypeptide. The Fc region may be a naturally occurring Fc region or may be altered to improve certain qualities such as treatment quality, cycle time, or reduced aggregation.

Useful modifications of protein therapeutics by fusion with the “Fc” domain of an antibody are discussed in detail in PCT Publication No. WO 00/024782. This document discusses connections to "vehicles" such as polyethylene glycol (PEG), dextran or Fc regions.

b. Fusion protein linker

When forming the fusion protein of the invention, although not necessary, a linker can be used. If present, the linker acts primarily as a spacer, so its chemical structure may not be important. The linker may consist of amino acids linked together by peptide bonds. In some embodiments of the invention, the linker consists of 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. In various embodiments, 1-20 amino acids are selected from amino acids glycine, serine, alanine, proline, asparagine, glutamine and lysine. In some embodiments, the linker consists of most amino acids that are stericly undisturbed, such as glycine and alanine. In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (such as Gly-Ala), or a combination of glycine and serine (such as Gly-Ser). Although linkers of 15 amino acid residues have been found to function particularly well for FGF21 fusion proteins, the present invention contemplates linkers of any length or composition.

Linkers described herein are exemplary and linkers comprising even longer and other residues are contemplated by the present invention. Non-peptide linkers are also contemplated by the present invention. For example, such as an alkyl linker can be used. These alkyl linkers are added by any non-stereoblocking group, including but not limited to lower alkyl (eg C1-C6), lower acyl, halogen (eg Cl, Br), CN, NH 2 or phenyl It may be substituted by. Exemplary non-peptide linkers are polyethylene glycol linkers, wherein the linker has a molecular weight of 100 to 5000 kD, for example 100 to 500 kD.

Chemically Modified Fusion Proteins

Chemically modified forms of the fusion proteins described herein, including, for example, truncated and variant forms of the FGF21 fusions described herein, can be prepared by one of ordinary skill in the art in view of the disclosure described herein. Such chemically modified fusion proteins are altered such that chemically modified mutants differ from unmodified mutants in the type or location of the molecule that is naturally attached to the mutant. Chemically modified mutants may include molecules formed by deletion of one or more naturally attached chemical groups.

In one embodiment, proteins of the invention can be modified by covalent attachment of one or more polymers. For example, the polymer selected is typically water soluble such that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Within the range of suitable polymers are mixtures of polymers. Preferably, for therapeutic use of the final product formulation, the polymer will be pharmaceutically acceptable. Non-water soluble polymers conjugated to the proteins of the invention also form an aspect of the invention.

Exemplary polymers can each have any molecular weight and can be branched or unbranched. The polymers each typically have an average molecular weight of about 2 kDa to about 100 kDa (the term "about" indicates that in the formulation of the water soluble polymer, some molecules will be larger than the mentioned molecular weight and some will be small). The average molecular weight of each polymer is preferably about 5 kDa to about 50 kDa, more preferably about 12 kDa to about 40 kDa, most preferably about 20 kDa to about 35 kDa.

Suitable water soluble polymers or mixtures thereof are suitable for derivatization of proteins, including N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycols (PEG) (mono- (C1-C10), alkoxy- or aryloxy-polyethylene glycols). PEG form used), monomethoxy-polyethylene glycol, dextran (such as low molecular weight, for example about 6 kD dextran), cellulose, or other carbohydrate based polymers, poly- (N-vinyl pyrrolidone) Polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), and polyvinyl alcohols. Also included in the present invention are bifunctional crosslinking molecules that can be used to prepare covalently attached FGF21 protein variant multimers. Also included in the present invention are FGF21 mutants covalently attached to polysialic acid.

Polysaccharide polymers are another type of water soluble polymer that can be used for protein modification. Thus, the fusion proteins of the invention fused to a polysaccharide polymer form an embodiment of the invention. Dextran is a polysaccharide polymer consisting of individual subunits of glucose predominantly linked by alpha 1-6 linkages. Dextran itself is available in a wide molecular weight range and is readily available at molecular weights from about 1 kD to about 70 kD. Dextran is a water soluble polymer suitable for use as a vehicle, alone or in combination with another vehicle (eg Fc). See for example International Publication No. WO 96/11953. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported. See, eg, European Patent Publication No. 0 315 456, which is incorporated herein by reference. The invention also includes the use of dextran from about 1 kD to about 20 kD.

In general, chemical modifications can be carried out under any suitable condition used to react the protein with the activated polymer molecule. Methods of preparing chemically modified polypeptides generally include (a) reacting a polypeptide with an activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions that the FGF21 protein variant is attached to one or more polymer molecules, And (b) obtaining the reaction product. Optimum reaction conditions will be determined based on known parameters and the desired result. For example, the greater the ratio of polymer molecules to protein, the greater the percentage of attached polymer molecules. In one embodiment of the invention, chemically modified FGF21 mutants may have a single polymer molecular moiety at the amino-terminus (see, eg, US Pat. No. 5,234,784).

In another embodiment of the invention, proteins of the invention may be chemically coupled to biotin. The biotin / protein of the invention is then bound to avidin to produce the tetravalent avidin / biotin / protein of the invention. In addition, the protein of the present invention is covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP), and the resulting conjugate is precipitated with anti-DNP or anti-TNP-IgM to form a 10-valent derivatized conjugate. Can be.

In general, conditions that can be alleviated or modulated by the administration of a chemically modified FGF21 mutant of the invention include those described herein for the protein of the invention. However, the chemically modified FGF21 mutants disclosed herein may have additional activity, enhanced or reduced biological activity, or other features such as increased or decreased half-life compared to unmodified FGF21 mutants.

Therapeutic Compositions and Administration of the Fusion Proteins

The invention also provides a therapeutic composition comprising at least one of the fusion proteins of the invention described herein in admixture with a pharmaceutically or physiologically acceptable formulation agent or pharmaceutically acceptable carrier selected to be suitable for the mode of administration. Combinations are specifically contemplated, for example, in terms of identifying proteins that exhibit enhanced properties.

In some embodiments, the therapeutic composition is prepared as an injection as a liquid solution or suspension; Solid forms suitable for solution or suspension in a liquid vehicle before injection can also be prepared. Liposomes are included within the definition of pharmaceutically acceptable carriers. Pharmaceutically acceptable salts such as inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate and the like; And salts of organic acids such as acetates, propionates, malonates, benzoates and the like can also be present in the pharmaceutical compositions. A detailed discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro, Lippincott, Williams, & Wilkins.

Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

Pharmaceutical compositions are for example used to modify, maintain or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation of the composition. Formulation materials may be contained. Suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite), buffers (such as borate, bicarbonate, tris-HCl, Citrate, phosphate or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy) Propyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavors and diluents, Emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, Forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, propylene glycol Or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tro Metamin; lecithin; cholesterol or tyloxapal), stability enhancers (such as sucrose or sorbitol), tonicity enhancers (such as alkali metal halides; preferably sodium chloride or potassium chloride; or mannitol sorbitol), delivery vehicles, diluents, excipients and / or Including but not limited to pharmaceutical adjuvants (For example, for any purpose a document incorporated by reference herein [Remington's Pharmaceutical Sciences (18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990)], and see his subsequent edition).

The optimal pharmaceutical composition will be determined by one skilled in the art depending, for example, on the intended route of administration, delivery format, and desired dosage (see, eg, Remington's Pharmaceutical Sciences, supra). Such compositions can affect the physical state, stability, rate of release in vivo, and rate of clearance in vivo of the fusion proteins of the invention.

Primary vehicles or carriers in pharmaceutical compositions may be aqueous or non-aqueous in nature. Vehicles or carriers suitable for injection, for example, may be water, physiological saline or artificial cerebrospinal fluid, possibly supplemented with other materials customary in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is an additional exemplary vehicle. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further comprise sorbitol or a suitable substitute. In one embodiment of the invention, the dual function pharmaceutical composition can be prepared for storage by mixing a selected composition having the desired purity with any formulating agent (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. have. In addition, the bifunctional protein product can be formulated as a lyophilisate using suitable excipients such as sucrose.

Pharmaceutical compositions containing the fusion proteins of the invention may be selected for parenteral delivery. Alternatively, the composition may be selected for inhalation or for delivery through the digestive tract, such as oral. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation component is present at an acceptable concentration at the site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically in the pH range of about 5 to about 8.

When parenteral administration is contemplated, the therapeutic composition for use in the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired dual function protein in a pharmaceutically acceptable vehicle. Particularly suitable vehicles for parenteral injection are sterile distilled water formulated as a sterile isotonic solution in which the dual function protein is suitably preserved therein. Still other formulations may be added to agents that provide controlled or sustained release of the product that can be delivered later via depot injection, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic or polyglycolic acid), beads or liposomes. Formulation of the molecule of interest. Hyaluronic acid may also be used, which may have the effect of promoting duration in circulation. Other means suitable for introducing a molecule of interest include an implantable drug delivery device.

In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the bifunctional protein of the present invention may be formulated as a dry powder for inhalation. Dual function protein inhalation solutions can also be formulated with propellants for aerosol delivery. In another embodiment, the solution can be sprayed. Pulmonary administration is further described in International Publication No. WO 94/20069, which describes pulmonary delivery of chemically modified proteins.

It is also contemplated that certain agents may be administered orally. In one embodiment of the invention, the fusion proteins of the invention administered in this manner may be formulated with or without the carriers conventionally used in the preparation of solid dosage forms such as tablets and capsules. For example, the capsule can be designed to release the active portion of the agent at a point in the gastrointestinal tract when bioavailability is at maximum and pre-systemic degradation is at a minimum. Additional agents may be included to promote uptake of the fusion proteins of the invention. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

Another pharmaceutical composition may comprise an effective amount of the fusion protein of the invention in admixture with a non-toxic excipient suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable vehicle, the solutions can be prepared in unit-dosage form. Suitable excipients include inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions comprising the fusion protein of the invention, such as formulations comprising the fusion protein of the invention in sustained- or controlled-delivery formulations, will be apparent to those skilled in the art. Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, biodegradable microparticles or porous beads and depot injections are known to those skilled in the art (e.g., of porous polymeric microparticles for delivery of pharmaceutical compositions). International Publication No. WO 93/15722 describing Controlled Release, and Wischke & Schwendeman, 2008, Int. J. Pharm. 364: 298-327 and Freiberg & Zhu, who discuss microsphere / microparticle preparation and use. 2004, Int. J. Pharm. 282: 1-18).

Further examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (US Pat. No. 3,773,919 and European Patent No. 0 058 481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56]), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem Tech. 12: 98-105), ethylene vinyl acetate (Langer et al., Supra) or poly-D-3-hydroxybutyric acid (European Patent No. 0 133 988). Sustained-release compositions may also include liposomes, which may be prepared by any of several methods known in the art. See, eg, Epstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82: 3688-92; And European Patent Nos. 0 036 676, 0 088 046 and 0 143 949.

Pharmaceutical compositions of the invention that can be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method may be performed before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions also generally are placed in a container equipped with a sterile access port, such as an intravenous solution bag or vial with a stopper that can be penetrated by a hypodermic needle.

Once the pharmaceutical composition has been formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored in ready-to-use form or in forms that require reconstitution prior to administration (eg, lyophilized form).

In a specific embodiment, the present invention relates to a kit for producing a single-dose dosage unit. The kit may each include both a first container with dried protein and a second container with aqueous formulation. Kits comprising single and multi-chamber pre-filled syringes (such as liquid syringes and lyo syringes) are also included within the scope of the present invention.

Dosage of fusion proteins and their administration

An effective amount of a pharmaceutical composition of the invention to be used therapeutically will vary depending on, for example, the therapeutic situation and the goal. Thus, those skilled in the art will understand in part depending on the molecule to which the appropriate dosage level is delivered for treatment, the indication using the fusion protein variant, the route of administration, and the body size (weight, body surface or organ size) and condition (age and overall health) of the patient. Will be recognized. Thus, the clinician can titrate the dosage and change the route of administration to obtain the optimal therapeutic effect. Typical dosages may range from about 0.1 μg / kg to about 100 mg / kg or more, depending on the factors mentioned above. In another embodiment, the dosage is 0.1 μg / kg to about 100 mg / kg; Or 1 μg / kg to about 100 mg / kg.

The frequency of administration will depend on the pharmacokinetic parameters of the dual function protein in the formulation used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the compositions can be administered as a single dose, as two or more dosed doses (with or without the same amount of the target molecule), or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosages is routinely made by those skilled in the art and falls within the area of work routinely performed by them. Appropriate dosages can be ascertained through the use of appropriate dose-response data.

The route of administration of the pharmaceutical composition may be in accordance with known methods, for example oral; Via injection by intravenous, intraperitoneal, intracranial (intraparenchymal), intraventricular, intramuscular, intraarterial, intraportal or intralesional routes; By sustained release systems, which can also be injected; Or by implantation device. If desired, the composition may be administered continuously by bolus injection, or infusion, or by an implantation device.

Alternatively or in addition, the composition may be administered topically via implantation of a membrane, sponge, or other suitable material on which the target molecule is absorbed or encapsulated thereon. If an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, delayed-release bolus, or continuous administration.

Therapeutic Uses of Fusion Proteins

Proteins of the invention can be used for the treatment, diagnosis, amelioration or prevention of many diseases, disorders or conditions, including but not limited to metabolic disorders. In one embodiment, the metabolic disorder treated is diabetes, eg type 2 diabetes. In another embodiment, the metabolic disorder is obesity. Other embodiments include type 1 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, hypertension, cardiovascular Diseases, acute myocardial infarction, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, disorders associated with severe inactivation mutations in the insulin receptor, gastric palsy and other metabolism Metabolic conditions such as disorders or disorders.

When applied, a disorder or condition, such as type 1 or type 2 diabetes or obesity, can be treated by administering to a patient in need thereof a FGF21 protein variant as described herein in a therapeutically effective amount. Administration can be performed orally as described herein, such as, for example, IV injection, intraperitoneal injection, intramuscular injection, or in tablet or liquid form. In most situations, the desired dosage may be determined by the clinician as described herein and may represent a therapeutically effective dose of the FGF21 mutant polypeptide. It will be apparent to those skilled in the art that the therapeutically effective dose of the FGF21 mutant polypeptide will depend in particular on the dosing regimen, unit dose of antigen administered, whether the nucleic acid molecule or polypeptide is administered in combination with other therapeutic agents, the immune status and health of the recipient. will be. The term “therapeutically effective dose” as used herein refers to a FGF21 that induces a biological or medicinal response including alleviation of symptoms of a disease or disorder being treated in a tissue system, animal or human being investigated by a researcher, physician, or other clinician. The amount of mutant polypeptide.

The present invention has been described in detail above, which will be more clearly understood by reference to the following examples which are included herein for illustrative purposes only and are not intended to limit the present invention.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology within the skill of the art. Such techniques are explained in detail in the literature. See, eg, Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); And Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); And Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989).

Example

Example 1: Preparation of FGF21 Variant Protein

Expression constructs for FGF21 V76: FGF21 variants are described by Achmuller et al. (2007) (Nature Methods 4: 1037-1043). Cloning into E. coli expression vector pET30a generated an in-frame fusion for the hexa-histidine tag followed by the N ^pro -EDDIE tag at the N-terminus (aa 33-209) of FGF21.

Expression and Purification of FGF21 V76: pET30a-His-N ^pro- EDDIE-FGF21 expression plasmid. The E. coli BL21 Star (DE3) competent cells (Invitrogen) were transformed. Growth was carried out overnight from a single colony of freshly transformed cells in 50 mL of Terrific Broth (TB) containing 50 μg / mL of kanamycin at 37 ° C. The pre-culture was transferred into 1 L TB medium containing kanamycin and incubated in a 37 ° C. baffle flask with shaking at 250 rpm. After 6 hours of incubation, the expression of FGF21 was induced by the addition of IPTG at a final concentration of 1 mM and the culture was grown overnight at 37 ° C. Cells are then harvested and 50 mL of ice cold lysis buffer; After resuspension in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, it was dissolved using a microfluidizer ™.

Inclusion bodies (IB) were precipitated by centrifugation at 30,000 × g at 4 ° C. for 1 hour. IB was washed with 50 mM Tris-HCl, pH 8, 150 mM NaCl, then 30 mL of lysis buffer; Dissolved in 10 mM Tris-HCl, pH 8, 100 mM NaH ₂ PO ₄ , 6M GnHCl. The dissolved IB was clarified by centrifugation at 30,000 × g at 25 ° C. for 1 hour. IB solution was loaded onto a 5 mL column of Ni-NTA high performance resin (GE Healthcare) equilibrated with lysis buffer. The protein bound to the resin was eluted by reducing the pH to 4.5. The eluate was conditioned by adjusting the pH and adding dithiothritol (DTT) at a concentration of 20 mM. The conditioned eluate was diluted with 1 L of refold buffer; Diluted slowly into 50 mM Tris-HCl, pH 8, 0.5 M arginine, 20 mM DTT, followed by incubation at 4 ° C. for 2 days. The diluted sample was concentrated and buffer-exchanged into 20 mM Tris-HCl, pH 9 using the ultrafiltration method. The concentrated sample was loaded onto a 10 mL column of Q sepharose fast flow resin (GE Healthcare) equilibrated with 20 mM tri-HCl, pH 9.

After washing the resin with equilibration buffer, the protein bound to the resin was eluted with 20 mM Tris-HCl, pH 9, 500 mM NaCl. The eluate was loaded on a 5 mL column of Ni-NTA high performance resin equilibrated with 20 mM Tris, pH 8.0, 50 mM imidazole, and the penetrant fraction containing FGF21 was collected and His cut off from the refolded FGF21 protein. -N ^pro fusion fragments and any ^uncut fusion proteins were removed. FGF21 fractions were treated with EndoTrap HD resin (Hyglos) equilibrated with 10 mM Tris, pH 8, 50 mM imidazole, 500 mM NaCl, 1 mM CaCl ₂ to reduce endotoxin levels. Low-endotoxin samples were dialyzed against PBS and then sterilized with a 0.22 μm filter. Purified FGF21 protein was rapidly-frozen in liquid nitrogen and stored at -80 ° C. Protein concentration was determined by absorbance at 280 nm using 9362 M-1 cm-1 as the molar extinction coefficient for FGF21. Protein purity and integrity were determined by HPLC, SDS-PAGE and liquid chromatography-mass spectrometry.

Cysteine PEGylation of FGF21 Variants: FGF21 variant V76 (R154C) has a tendency to dimerize through engineered cysteine; Accordingly, protein solution (typically 5 mg / ml in Tris buffer) was gently reduced to 5 mM mercaptoethylamine for 30 minutes on ice and immediately desalted in 20 mM Tris, pH 7 before PEGylation. The newly reduced protein (typically 3 mg / ml) was then subjected to 1.5 equivalents of 40 kDa branched maleimido-PEG reagent (NOF, catalog # GL2-400MA from Sunbright series) for 3 hours on ice. PEGylation immediately. PEGylated proteins were finally purified by anion exchange chromatography (MonoQ) in a total yield of about 25%.

Expression constructs for Fc-FGF21 fusion variants: cDNA for human FGF21 variant coding amino acids 33-209, followed by a leader peptide (immunoglobulin kappa-chain) directing the secretion of the protein followed by an Fc domain and a shorter linker Cloned in-frame with the N-terminal sequence downstream of the mammalian expression vector of the cytomegalovirus (CMV) promoter.

Expression and Purification of Fc-FGF21 Variants: Fc-FGF21 variant proteins were expressed in HEK293T cells (American Type Culture Collection). Cells were grown in suspension culture at 37 ° C., 8% CO ₂ in Freestyle 293 expression medium (Invitrogen, Catalog # 12338-018) until the day of transfection. Cells were centrifuged at 1000 × g for 7 minutes in a shaking bucket rotor and counted using an automatic cell counter. The cells were diluted to a final concentration of 1.4 × 10 ⁶ cells / mL in 900 mL of Freestyle 293 medium and placed in a 3 L non-baffle flask (Corning, catalog # 431252). Cells were transfected using a mixture of polyethyleneimine (PEI) and plasmids as follows. Add 3 mL of sterile 1 mg / ml stock, 3 mL of MW25,000, PEI (Alfa Aesar, Catalog # 43896) to 50 mL of Freestyle 293 medium, mix gently and 25 ° C. for 5 minutes. Incubated at. At the same time, 1 mg of endotoxin-free plasmid was added to 50 mL Freestyle 293 medium and sterile filtered using a 0.22 uM filter. The PEI mixture was then added to sterile filtered DNA, mixed gently and allowed to incubate at 25 ° C. for 10 minutes. The PEI-plasmid mixture was then added to a 3 L flask containing diluted HEK 293T cells and placed in a 125 RPM, 37 ° C., 8% CO ₂ shake incubator.

On day 6 after transfection, supernatants were harvested by centrifuging the cells at 2000 × g for 10 minutes. The supernatant was further purified by filtration through a 0.8 / 0.2 uM filter (Pall Corporation, Catalog # 4628).

Batch purification of FGF21 protein was added directly to the clarified supernatant with 1 mL of recombinant Protein A Sepharose Fast Flow (GE, Catalog # 17-5138-03) per 20 mg of the protein expected to be purified and rotated gently at 4 ° C. Incubation for 1 h at. The supernatant mixture was then poured onto a disposable poly-preparative chromatography column (Bio-Rad, catalog # 731-1550) and the penetrant was discarded. Residual beads were washed with 5 column volumes of DPBS, pH 7.4 (Invitrogen, Catalog # 14190-144). Elution of the protein from the Protein A beads was done by adding 20 column volumes of 50 mM sodium citrate buffer, pH 3.0. Elution buffer was neutralized by the addition of 20% Tris-HCL buffer, pH 9.0. Size exclusion chromatography was performed as a second polishing step by flowing Protein A batch purified material onto a High Load 26/600 Superdex 200 pg column (GE, Catalog # 28-9893-36). . Purified protein yield was quantified by A280. SDS-Page was run to verify purity and molecular weight. Endotoxin levels were quantified using the Endosafe PTS system (Charles River Labs).

Example 2: Determination of FGF21 dependent 2-deoxyglucose (2-DOG) uptake

FGF21 stimulates glucose-absorption in mouse 3T3-L1 adipocytes in the presence and absence of insulin and postprandial and fasting blood glucose, triglycerides, in ob / ob and db / db mice and 8-week-old ZDF rats in a dose-dependent manner, and It has been shown to reduce glucagon levels and thus provides a basis for the use of FGF21 as a therapy for treating diabetes and obesity (see, eg, Patent Publication WO03 / 011213 and Kharitonenkov et al., (2005) Jour. Of Clinical Invest. 115: 1627-1635). FGF21 was also observed to stimulate tyrosine phosphorylation of FGFR-1 and FGFR-2 in 3T3-L1 adipocytes.

3T3-L1 fibroblasts were purchased from ATCC (Catalog # CL173). Cells were grown to 150 cm Petri-Dish to confluent growth and in DMEM (Invitrogen, Catalog # 11995065) with high glucose supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin for an additional 4 days. Maintained. Cells were then incubated with 4 μg / mL insulin (Sigma, Catalog # I-5500), 115 μg / mL IBMX (Sigma, Catalog # I5879) and 0.0975 μg / mL dexamethasone (Sigma, Catalog # D1756) for 3 days. After differentiation in the supplemented medium, the differentiation medium was replaced with complete DMEM. One plate of differentiated 3T3-L1 adipocytes was seeded in four 96-well plates the day after medium change.

Adipocytes were then treated overnight with FGF21-WT and FGF21 variant proteins (see Table 2 for variant listing; using typical concentration ranges from 30 pM to 100 nM) in complete medium. Adipocytes treated with FGF21 samples were treated with 50 μL / well of KRH buffer (0.75% NaCl; 0.038% KCl; 0.0196% CaCl ₂ ; 0.032% MgSO ₄ ; 0.025M HEPES, pH 7.5; 0.5% BSA; 2 mM for 2 hours. Serum depleted) in sodium pyruvate). 1 μL (final concentration 5 μg / ml) cytokalcin B was added to the wells for the blanks for 15 minutes. [3H] -2-DOG (20.6 mCi / mmol, 1 mCi / ml) is diluted 1:20 in 5.1 mM cold 2-DOG, 1 μL of diluted 2-DOG is added per well and cells are 5 Incubate for minutes. Cells were washed three times with 100 μL / well of KRH buffer. 40 μL / well of 1% SDS was added to the cells and the cells shaken for at least 10 minutes. 200 μL / well scintillation fluid was added and the plate shaken overnight and read in a beta-microplate reader. Values obtained from all rows / columns treated with cytocalin B were averaged and subtracted from all other values. Data was analyzed by GraphPad Prism software and the results are summarized in Table 2. Fc-FGF21 fusion variants V101, V103 and V188 were superior to PEGylated FGF21 variant V76 for induction of 2-deoxyglucose uptake by mouse 3T3L1 adipocytes.

Example 3: pERK In Cell Western (ICW) Assay.

HEK293 cells stably transfected with human β-cloto were incubated in DMEM high glucose, 10% FBS, 1% PS and 600 ng / ml G418, and poly-D-lysine coated 96-well plates (BD Biosciences BD bioscience), catalog # 356640), seeded overnight at 30,000 cells / well. Cells were depleted of serum in DMEM high glucose, 0.5% BSA and 10 mM HEPES for 4 hours. WT FGF21 and FGF21 variants (see Table 3 for a list of variants) were diluted in various concentrations (using typical concentration ranges from 100 pM to 300 nM) in depleting medium. Cells were stimulated with FGF21 for 10 minutes. After stimulation of FGF21 or FGF21 variant protein, the medium is aspirated from the wells, the cells are washed once with 100 μL of cold PBS and then fixed at 100 μl of 4% formaldehyde for 15 minutes at room temperature followed by 100 μL ice cold methanol Incubated for an additional 10 minutes.

After fixation, cells were washed four times with 0.3% Triton X-100 in PBS for 5 minutes each. 150 μL Odyssey blocking buffer was added to the permeated cells at room temperature for 1.5 hours. Dilute phospho-ERK (pERK) antibodies to a concentration of 0.17 μg / ml (1: 200 dilution or suggested dilution), and total-ERK (tERK) antibodies to a concentration of 2.2 μg / ml in odyssey blocking buffer (1: 200 dilution or indicated dilution). 50 μL was added to all wells except one column treated only with secondary antibody to normalize against background. The plate was covered with a wet paper towel and lid, prevented evaporation and incubated overnight at 4 ° C.

The primary antibody was then aspirated and cells were washed four times with 0.3% Tween 20 in PBS for 5 minutes each. During washing, the secondary antibody reaction mixture was subjected to 1: 1000-dilution (or suggested dilution) goat anti-mouse Alexa 680 and 1: 1000-dilution (or indicated dilution) IRDye800 goat anti-rabbit. Prepared in odyssey blocking buffer containing the antibody. When the wash is complete, 40 μL of the reaction mixture is added to each well. The plate was covered with a black lid to shade secondary antibody and the plate was incubated for 1 hour at room temperature on a shaker. Finally, the cells were washed again four times with 0.3% Tween 20 in PBS for 5 minutes each, followed by the LI-COR Bioscience Odyssey Infrared Imaging System (Li-Cor Bioscience, Lincoln, Nebraska, USA). Sheath) was scanned into 700 nm (red) and 800 nm (green) channels. Alexa 680 stained tERK with near infrared fluorescence (emission wavelength 668 nm), while AIDI800 stained pERK with green fluorescence (emission wavelength 800 nm). Values obtained from all rows / columns treated with secondary antibodies only to remove fluorescent backgrounds were averaged and subtracted from all other values obtained from plates. The value for pERK in each well was divided by the value of tERK to normalize the amount of pERK present in each sample. The data was analyzed by GraphPad Prism software and the results are summarized in Table 2. Fc-FGF21 fusion variants V101, V103 and V188 were superior to PEGylated FGF21 variants V76 in this ERK phosphorylation assay.

Table 2: Overview of ERK Phosphorus Cell Western and Mouse 3T3L1 Adipocyte Glucose Uptake Assay Results

Example 4: In Vivo Testing of FGF21 Variants

ob / ob mice are a mouse model for type 2 diabetes. Mice lack functional leptin and are characterized by hyperglycemia, insulin resistance, bulimia, hepatic steatosis and obesity. Male ob / ob mice (10-13 weeks old) were used to determine the effect of the following PEGylated FGF21 variant V76 and Fc-FGF21 fusion variants V101, V103 and V188 on blood glucose.

FGF21 variant or PBS vehicle was administered subcutaneously at 1 mg / kg (V101, V103 and V188) twice daily or 12 days twice weekly (4 total doses) or 5 mg / kg V76. On day 1 of the study, tail blood glucose and body weight were measured and mice were distributed among groups with different groups (n = 8 per group) in which mean glucose and body weight matched. Blood glucose was measured using a blood glucose meter (OneTouch). Plasma insulin was measured on day 1, before day 12, and 24 hours after the last dose. The results of these studies are summarized in Table 5.

The results of these studies are summarized in Table 3 and FIGS. 1-3. Fc-FGF21 fusion variants V101, V103 and V188 were superior to PEGylated FGF21 variant V76 at all endpoints and 5 times lower doses measured in these studies.

Table 3. Changes in plasma glucose, insulin, weight gain (BW), vehicle in liver TG / lipids by FGF21 variant during 12-day study in ob / ob mice.

Example 5: Pharmacokinetics of FGF21 Fusion Variants in Mice

C57BL / 6J mice were IV injected with 1 mg / kg test material and blood collected at various time points over 16 days (384 hours) to determine the pharmacokinetic profiles of Fc-FGF21 fusion variants V101, V103 and V188. Blood samples were collected from the submandibular and orbital veins in EDTA-coated microtainer tubes. Approximately 50 μL of blood was collected at each time point to obtain ˜25 μL of plasma.

384-well plates were coated overnight at room temperature with 2 μg / mL of anti-human Fc-gamma goat polyclonal antibody (30 μL / well), followed by casein-based diluent (100 μL / well) at room temperature for 2 hours. Blocking by, the plasma concentration of the test substance was measured by ELISA. Diluted samples, standards and controls were added to the plate (30 μL / well) and incubated for 2 hours at room temperature. After removing the sample, the wells were washed three times with phosphate-based washes (100 μL / well). The detection antibody, the HRP-labeled version of the capture antibody, was added to the plate (30 μL / well) and incubated for 1 hour at room temperature. The plates were washed three more times with phosphate-based wash solution (100 μL / well), then the chemiluminescent substrate was added (30 μL / well) and plate luminescence was read within 5 minutes using an appropriate plate reader. As shown in FIGS. 4A and 4B, the Fc-FGF21 fusion variant had a much longer plasma half-life compared to the Fc-FGF21 fusion (FIG. 4A) and PEGylated FGF21 variant V76 (FIG. 4B) known in the art.

Serum levels of the Fc-FGF21 test substance were verified by Western blot for comparison with the levels measured by ELISA to ensure that full-length Fc-FGF21 variants but not Fc alone were detected in ELISA. 2 uL of mouse serum is combined with 2.5 uL of 4 × loading buffer, 1 uL of 10 × denaturant and 4 uL of dH ₂ O, heated to 95 ° C. for 5 minutes, and loaded onto a 4-12% gradient polyacrylamide gel. And electrophoresis for 1 hour at 100 volts (constant voltage). Samples were transferred to nitrocellulose filter paper by Western blot using iBlot system (Invitrogen Catalog # IB1001, run time of 7 minutes). The nitrocellulose filter was blocked with 30 mL Rockland blocking solution (catalog # MB-070) and diluted 1: 2000 goat anti-FGF21 primary antibody (R & D systems, catalog # BAF2539) and The probes were probed according to the Snap Iblot System Protocol with fluorescently labeled streptavidin (Licor, catalog # 926-68031) as a secondary diluted 1: 10000. Protein levels were imaged at 700 nm on the Licor Odyssey system and compared to 2 nM control V101 run on the same gel. As shown in FIG. 4C, full length Fc-FGF21 variants V101, V103 and V188 were detectable using Western blot using anti-FGF21 antibody over 15 days from mouse serum in pharmacokinetic studies.

Example 6: Fc-FGF21 Fusion Variants V101, V103 and V188 are Thermodynamically Very Stable

Proteins can be unfolded at specific temperature ranges. The temperature of protein unfolding is an inherent parameter for explaining the thermal stability of proteins. Differential scanning calorimetry (DSC) is used to detect the non-folding temperature of the protein. This characteristic temperature is described as the melting temperature (Tm), which is the peak temperature during protein unfolding.

The original protein sample was diluted in PBS to a concentration of ˜1 mg / ml (0.5 mg / ml to 1.2 mg / ml) for a total volume of 0.5 ml. 0.4 ml / well aliquots of diluted protein samples, standards, PBS and deionized water were added to the DSC 96-well plates. The plate was then covered by sealing. Samples were analyzed in a 96 well differential scanning calorimeter from MicroCal. The temperature was scanned at 10-110 ° C. at a rate of 1 degree / minute.

As shown in FIG. 4D, the melting temperatures of the FGF21 variants V101, V103 and V188 were very high. This is in contrast to the lower melting temperatures (not shown) of FGF21 variant V76 and wild type FGF21. We saw improved stability of V101, V103 and V188 as a result of the specific addition of the second disulfide bonds from the novel Q55C and G148C mutations. This type of thermodynamic stability is known to protect proteins from proteolysis and can also be interpreted as a significantly extended in vivo stability and improved pharmacokinetic profile illustrated by the data of FIGS. 4B and 4C.

                                SEQUENCE LISTING <110> Boettcher, Brian       Daniels, Doug       Caplan, Shari       Hamamatsu, Norio       Weldon, Stephen       Licht, Stuart <120> FUSION PROTEINS FOR TREATING METABOLIC   DISORDERS <130> PAT054783 <150> 61/539280 <151> 2011-09-26 <160> 13 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 209 <212> PRT <213> Homo sapiens <400> 1 Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser  1 5 10 15 Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro             20 25 30 Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr         35 40 45 Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg     50 55 60 Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu 65 70 75 80 Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val                 85 90 95 Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly             100 105 110 Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu         115 120 125 Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu     130 135 140 His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150 155 160 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu                 165 170 175 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp             180 185 190 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala         195 200 205 Ser      <210> 2 <211> 940 <212> DNA <213> Homo sapiens <400> 2 ctgtcagctg aggatccagc cgaaagagga gccaggcact caggccacct gagtctactc 60 acctggacaa ctggaatctg gcaccaattc taaaccactc agcttctccg agctcacacc 120 ccggagatca cctgaggacc cgagccattg atggactcgg acgagaccgg gttcgagcac 180 tcaggactgt gggtttctgt gctggctggt cttctgctgg gagcctgcca ggcacacccc 240 atccctgact ccagtcctct cctgcaattc gggggccaag tccggcagcg gtacctctac 300 acagatgatg cccagcagac agaagcccac ctggagatca gggaggatgg gacggtgggg 360 ggcgctgctg accagagccc cgaaagtctc ctgcagctga aagccttgaa gccgggagtt 420 attcaaatct tgggagtcaa gacatccagg ttcctgtgcc agcggccaga tggggccctg 480 tatggatcgc tccactttga ccctgaggcc tgcagcttcc gggagctgct tcttgaggac 540 ggatacaatg tttaccagtc cgaagcccac ggcctcccgc tgcacctgcc agggaacaag 600 tccccacacc gggaccctgc accccgagga ccagctcgct tcctgccact accaggcctg 660 ccccccgcac tcccggagcc acccggaatc ctggcccccc agccccccga tgtgggctcc 720 tcggaccctc tgagcatggt gggaccttcc cagggccgaa gccccagcta cgcttcctga 780 agccagaggc tgtttactat gacatctcct ctttatttat taggttattt atcttattta 840 tttttttatt tttcttactt gagataataa agagttccag aggagaaaaa aaaaaaaaaa 900 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 940 <210> 3 <211> 181 <212> PRT <213> Homo sapiens <400> 3 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val  1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His             20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser         35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln     50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg                 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His             100 105 110 Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro         115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro     130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser                 165 170 175 Pro Ser Tyr Ala Ser             180 <210> 4 <211> 546 <212> DNA <213> Homo sapiens <400> 4 caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60 ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 120 gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 180 ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240 gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 300 gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360 aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 420 ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 480 ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 540 tcctga 546 <210> 5 <211> 28 <212> PRT <213> Homo sapiens <400> 5 Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser  1 5 10 15 Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala             20 25 <210> 6 <211> 15 <212> DNA <213> Homo sapiens <400> 6 ggggsggggs ggggs 15 <210> 7 <211> 406 <212> PRT <213> Homo sapiens <400> 7 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly  1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly Lys Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln 225 230 235 240 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala                 245 250 255 His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln             260 265 270 Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile         275 280 285 Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp     290 295 300 Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe 305 310 315 320 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala                 325 330 335 His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp             340 345 350 Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro         355 360 365 Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp     370 375 380 Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg 385 390 395 400 Ser Pro Ser Tyr Ala Ser                 405 <210> 8 <211> 419 <212> PRT <213> Homo sapiens <400> 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly  1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln                 245 250 255 Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu             260 265 270 Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu         275 280 285 Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu     290 295 300 Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu 305 310 315 320 Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu                 325 330 335 Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu             340 345 350 Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro         355 360 365 Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu     370 375 380 Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser 385 390 395 400 Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser                 405 410 415 Tyr ala ser              <210> 9 <211> 177 <212> PRT <213> Homo sapiens <400> 9 Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr  1 5 10 15 Leu Tyr Thr Asp Asp Ala Gln Glu Thr Glu Ala His Leu Glu Ile Arg             20 25 30 Glu Asp Gly Thr Val Gly Gly Ala Ala His Gln Ser Pro Glu Ser Leu         35 40 45 Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val     50 55 60 Lys Thr Ser Arg Phe Leu Cys Gln Lys Pro Asp Gly Ala Leu Tyr Gly 65 70 75 80 Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu                 85 90 95 Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu             100 105 110 His Leu Pro Gly Asn Arg Ser Pro His Cys Asp Pro Ala Pro Gln Gly         115 120 125 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu     130 135 140 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp 145 150 155 160 Pro Leu Ala Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala                 165 170 175 Ser      <210> 10 <211> 406 <212> PRT <213> Homo sapiens <400> 10 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly  1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly Lys Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln 225 230 235 240 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala                 245 250 255 His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln             260 265 270 Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile         275 280 285 Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp     290 295 300 Gly Thr Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe 305 310 315 320 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala                 325 330 335 His Gly Leu Pro Leu His Leu Pro Cys Asn Arg Ser Pro His Arg Asp             340 345 350 Pro Ala Ser Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro         355 360 365 Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp     370 375 380 Val Gly Ser Ser Asp Pro Leu Ala Met Val Gly Gly Ser Gln Ala Arg 385 390 395 400 Ser Pro Ser Tyr Ala Ser                 405 <210> 11 <211> 406 <212> PRT <213> Homo sapiens <400> 11 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly  1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly Lys Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln 225 230 235 240 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala                 245 250 255 His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln             260 265 270 Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile         275 280 285 Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Lys Pro Asp     290 295 300 Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe 305 310 315 320 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala                 325 330 335 His Gly Leu Pro Leu His Leu Pro Cys Asn Arg Ser Pro His Arg Asp             340 345 350 Pro Ala Ser Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro         355 360 365 Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp     370 375 380 Val Gly Ser Ser Asp Pro Leu Ala Met Val Gly Gly Ser Gln Ala Arg 385 390 395 400 Ser Pro Ser Tyr Ala Ser                 405 <210> 12 <211> 419 <212> PRT <213> Homo sapiens <400> 12 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly  1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln                 245 250 255 Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala His Leu Glu             260 265 270 Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu         275 280 285 Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu     290 295 300 Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Lys Pro Asp Gly Ala Leu 305 310 315 320 Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu                 325 330 335 Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu             340 345 350 Pro Leu His Leu Pro Cys Asn Arg Ser Pro His Arg Asp Pro Ala Ser         355 360 365 Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu     370 375 380 Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser 385 390 395 400 Ser Asp Pro Leu Ala Met Val Gly Gly Ser Gln Ala Arg Ser Pro Ser                 405 410 415 Tyr ala ser              <210> 13 <211> 419 <212> PRT <213> Homo sapiens <400> 13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly  1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln                 245 250 255 Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala His Leu Glu             260 265 270 Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu         275 280 285 Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu     290 295 300 Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Thr Leu 305 310 315 320 Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu                 325 330 335 Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu             340 345 350 Pro Leu His Leu Pro Cys Asn Arg Ser Pro His Arg Asp Pro Ala Ser         355 360 365 Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu     370 375 380 Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser 385 390 395 400 Ser Asp Pro Leu Ala Met Val Gly Gly Ser Gln Ala Arg Ser Pro Ser                 405 410 415 Tyr ala ser             

Claims (31)

A fusion protein comprising a FGF21 variant and an Fc region, wherein the FGF21 variant comprises mutations of Q55C, R105K, G148C, K150R, P158S, S195A, P199G and G202A for the full length hFGF21 sequence. A fusion protein comprising the FGF21 variant and the Fc region and comprising the amino acid sequence of SEQ ID NO: 11. The fusion protein of claim 1, wherein the FGF21 variant is fused to said Fc region by a GS linker. The fusion protein of claim 1, wherein the Fc region is a modified Fc fragment having a LALA mutation. The fusion protein of claim 1, comprising the amino acid sequence of SEQ ID NO: 12. The fusion protein of claim 1, wherein the Fc region is linked to a FGF21 variant via a linker. 7. The fusion protein of claim 6, wherein the linker is 1-20 amino acids in length. 8. The fusion protein of claim 6 or 7, wherein the linker comprises glycine and serine residues. 8. The fusion protein of any one of claims 1, 3, 6 and 7, wherein the Fc region of the fusion protein is a modified Fc fragment. A blood glucose lowering, lowering insulin level, lowering triglyceride level, lowering cholesterol level, lowering liver lipid level, lowering liver triglyceride level, weight loss, in a patient comprising the fusion protein according to any one of claims 1-7. Pharmaceutical compositions for use in achieving one or more biological activities selected from the group consisting of improving glucose tolerance and improving insulin sensitivity. 11. The patient of claim 10, wherein the patient is associated with obesity, type 1 and type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, diabetes complications, gastric palsy, severe inactivation mutations in the insulin receptor. A pharmaceutical composition comprising one or more disorders selected from the group consisting of associated disorders, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and other metabolic disorders. The pharmaceutical composition of claim 11, wherein the disorder is obesity, type 2 diabetes, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or hyperglycemia. A polynucleotide encoding the fusion protein of any one of claims 1 to 7. A vector comprising the polynucleotide of claim 13. A vector comprising the polynucleotide of claim 13, or
The polynucleotide of claim 13
Host cell comprising a. A method of producing a fusion protein, comprising expressing the fusion protein using the host cell of claim 15. The method of claim 16, further comprising purifying the fusion protein. A pharmaceutical composition for use in a method of treating a metabolic disorder in a patient, comprising the fusion protein according to any one of claims 1 to 7. The pharmaceutical composition of claim 18, wherein the metabolic disorder is obesity, diabetes, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) or hyperglycemia. The pharmaceutical composition of claim 19, wherein the metabolic disorder is type 2 diabetes. The pharmaceutical composition of claim 19, wherein the metabolic disorder is non-alcoholic fatty liver disease (NAFLD). The pharmaceutical composition of claim 19, wherein the metabolic disorder is nonalcoholic steatohepatitis (NASH). delete delete delete delete delete delete delete delete delete

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